The EMBO Journal

Published by EMBO Press
Online ISSN: 1460-2075
Autoradiography obtained from immunoblots of different tissue homogenates of the rat after labeling with affinity-purified serum antibodies for synaptophysin (upper part) and synaptobrevin (lower part) as described in the legend to Figure 3. Aliquots of protein (10 i.g/lane) were separated. Lanes 1 and 2, cortex; lane 3, cerebellum; lane 4, skeletal muscle; lane 5, liver; lane 6, kidney. In addition to the tissues shown, the following rat tissue homogenates were examined and found to be negative: parotid gland, spleen and heart muscle. To increase sensitivity, exposure time for synaptobrevin was six-times longer than for synaptophysin (lane 2-6). Lane 1, identical exposure time for both proteins to show the relative strength of signals at saturating antibody concentrations. 
A protein with an apparent mol. wt of 18,000 daltons (synaptobrevin) was identified in synaptic vesicles from rat brain. Some of its properties were studied using monoclonal and polyclonal antibodies. Synaptobrevin is an integral membrane protein with an isoelectric point of approximately 6.6. During subcellular fractionation, synaptobrevin followed the distribution of small synaptic vesicles, with the highest enrichment in the purified vesicle fraction. Immunogold electron microscopy of subcellular particles revealed that synaptobrevin is localized in nerve endings where it is concentrated in the membranes of virtually all small synaptic vesicles. No significant labeling was observed on the membranes of peptide-containing large dense core vesicles. In agreement with these results, synaptobrevin immunoreactivity has a widespread distribution in nerve terminal-containing regions of the central and peripheral nervous system as shown by light microscopy immunocytochemistry. Outside the nervous system, synaptobrevin immunoreactivity was found in endocrine cells and cell lines (endocrine pancreas, adrenal medulla, PC12 cells, insulinoma cells) but not in other cell types, for example smooth muscle, skeletal muscle and exocrine pancreas. Thus, the distribution of synaptobrevin is similar to that of synaptophysin, a well-characterized membrane protein of small vesicles in neurons and endocrine cells.
Effect of pre-treatment with PBt2 on the phosphorylation of 80 K in cell-free extracts. Quiescent cultures of Swiss 3T3 cells were exposed to 200 ng/ml of PBt2 for 40 h. After this time, detergent-solubilized extracts from the same number of control (C) and PBt2-treated (PT) dishes were prepared. The phosphorylation assay was performed with 0.5 mM EGTA, 0.45 mM CaC12, at 30°C for 15 s in the absence (-) or the presence (+) of 100 ng/ml PBt2 and 80 yg/ml PS. measured in cell-free extracts (Rodriguez-Pena and Rozengurt, 1984), and desensitizes the cells to further biological effects of PBt2 (Collins and Rozengurt, 1984; Rozengurt et al., 1983a, 1984, 1985). Here we show that prolonged treatment with PBt2 of intact Swiss 3T3 cells prior to the preparation of the cell-free extracts, completely prevented the stimulation of 80 K phosphorylation caused by the addition of protein kinase C activators (i.e., PS, Ca2+ and PBt2). This result is in accord with the disappearance of 80 K phosphorylation in intact cells and further substantiates the proposition that the phosphorylation of 80 K reflects the activation of protein kinase C. If prolonged pre-treatment with PBt2 selectively reduces protein kinase C, it should be possible to restore the ability of the cell-free system to phosphorylate 80 K by adding exogenous protein kinase C. The results presented in Figure 7 are in line with this prediction. This finding has several important implications: (i) it provides further evidence that 80 K phosphorylation is promoted by protein kinase C; (ii) it rules out the possibility that the failure of PS and PBt2 to stimulate 80 K phosphorylation in cell-free extracts prepared from PBt2-treated cells is due merely to a low rate of turnover of the 80 K phosphoprotein generated during the early stages of the treatment with PBt2, and (iii) it strongly suggests that protein kinase C and 80 K are separate proteins, although direct confirmation of this hypothesis will require further experimental work. Recent evidence demonstrates that activation of protein kinase C by diacylglycerol and phorbol esters acts as a mitogenic signal for quiescent 3T3 cells (Rozengurt et al., 1984, 1985). Hence the stimulation of this phosphotransferase system may play a fundamental role in effecting the proliferative response elicited by certain growth factors. A crucial step to evaluate this hypothesis  
Activation of the endogenous Ca2+-activated phospholipid-dependent protein kinase (protein kinase C) by Ca2+, phosphatidylserine (PS) and phorbol dibutyrate (PBt2) in detergent-solubilized extracts of Swiss 3T3 cells resulted in a very rapid increase (detectable within seconds) in the phosphorylation of an 80 000 mol. wt. protein (termed 80 K). Neither cyclic AMP nor Ca2+ had any effect on 80 K phosphorylation. The 80 K phosphoproteins generated after activation of protein kinase C, both in cell-free conditions and in intact fibroblasts, are identical as judged by one and two-dimensional polyacrylamide slab gel electrophoresis and peptide mapping. Prolonged treatment of cells with phorbol esters causes a selective decrease in protein kinase C activity and prevents the stimulation of 80 K phosphorylation in intact fibroblasts. We now show that extracts from PBt2-treated cultures fail to stimulate 80 K phosphorylation after the addition of the protein kinase C activators. This effect was due to the lack of protein kinase C activity since the addition of exogenous protein kinase C from mouse brain stimulated 80 K phosphorylation in both control and PBt2-treated preparations. The 80 K phosphoprotein generated by activation of endogenous and exogenous protein kinase C yielded similar phosphopeptide fragments after peptide mapping by limited proteolysis. We conclude that the detection of changes in the phosphorylation of 80 K provides a useful approach to ascertain which extracellular ligands activate protein kinase C in intact cells.
Purification of hsp9O from rabbit reticulocyte lysate, HeLa cell extract and L-929 cell extract. The purification procedure is described in Materials and methods. The figure shows a photograph of a Coomassie brilliant blue stained gel after different stages of purification of 5 ml of (b)-(f) rabbit reticulocyte lysate, (h)-(I) HeLa cytoplasmic extract and (n)-(r) L-929 cytoplasmic extract. Tracks (b), (h) and (n) are of the original extract; (c), (i) and (o) are S1OO supernatants; (d), (j) and (p) are the 35% ammonium sulphate supernatants; (e), (k) and (q) are the pool of fractions containing hsp9O after S200 gel filtration; (f), (I) and (r) are the pool of fractions containing hsp9O after anion exchange chromatography. Tracks (a), (g), (m) and (s) show mol. wt. markers. 
Double-stranded DNA (dsDNA) induces the transfer of phosphate from ATP to several proteins in extracts of widely divergent eukaryotic cells. Extracts of HeLa cells, rabbit reticulocytes, Xenopus eggs and Arbacia eggs all show dsDNA-dependent protein phosphorylation. The mechanism is specific for dsDNA and will not respond to either RNA or single-stranded DNA. One of the proteins which is phosphorylated in response to dsDNA has a subunit mol. wt. of 90 000 and has been identified as a heat-shock protein (hsp90). Although mouse cell extracts were shown to contain hsp90, they failed to show a dsDNA-dependent protein phosphorylation. The observation that dsDNA can modulate the phosphorylation of a set of proteins raises the possibility that dsDNA may play a role as a cellular regulatory signal.
Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo.
Components of the ubiquitin conjugating system were purified from human placenta by covalent affinity chromatography on ubiquitin sepharose. In contrast to E2 preparations obtained from rabbit reticulocytes and erythrocytes or Saccharomyces cerevisiae, the placental E2 preparation lacks E2(Mr = 14,000) and E2(Mr = 20,000) which are both unique in catalysing the ligase-independent transfer of ubiquitin to histones. A novel technique was employed to detect ubiquitin carrier function of the E2 proteins after SDS-electrophoresis and blotting to nitrocellulose. A cDNA of E2(Mr = 17,000) was isolated from a human cDNA library by screening with a degenerate oligonucleotide whose sequence was based on a partial amino acid sequence obtained from an E2(Mr = 17,000) peptide. Sequence analysis demonstrated an identity of 69% in the primary sequence of human E2(Mr = 17,000) and the protein encoded by the yeast DNA repair gene RAD6, which was recently shown to be an E2 species in yeast. Such a high degree of similarity between the human E2(Mr = 17,000) and the yeast DNA repair enzyme is suggestive of important common structural constraints or roles in addition to ubiquitin carrier activity, since in yeast this function itself is not necessarily dependent on high conservation of primary structure.
Electron micrographs of alkaline extracted and DTT or 3-mercaptoethanol reduced ACh-receptor rich membrane. (a) Freeze-etched view of pH 11 treated membrane. Note the reticular aspect of the distribution of the receptor rosettes at the membrane surface. (b) and (c) Freeze-etched aspect of a membrane fraction reduced by 20 mM fl-mercaptoethanol: no comparable reticulation occurred upon freezing. (d) Negatively-stained membranes reduced by DTT after fusion with phosphatidylcholine vesicles. Clusters or file of rosettes are visible. Comparable images are obtained with unreduced native membrane. (e) x 160 000. 
The rotational diffusion of the acetylcholine (ACh) receptor in subsynaptic membrane fragments from Torpedo marmorata electric organ was investigated with a spin-labelled alpha-bungarotoxin. A toxin with two spin labels was first synthesized; the conventional electron spin resonance spectrum (e.s.r.) of this toxin bound to the receptor indicated: (1) a complete immobilization of the probes; and (2) a strong spin-spin interaction that was not, or barely, seen in solution. The modification of the degree of spin-spin interaction is taken as an indication of a toxin conformational change accompanying its binding to the ACh-receptor. To avoid spin-spin interaction a single-labelled toxin was made and used to follow the rotational diffusion of the receptor by saturation transfer e.s.r. (ST-e.s.r.). With native membranes a high immobilization of the ACh-receptor was noticed. Reduction of the membranes by dithiothreitol had little effect on this motion. Only extraction of the 43 000 protein(s) by pH 11 treatment was able to enhance the rotational diffusion of the ACh-receptor protein (rotational correlation time by ST-e.s.r. in the 0.5 - 1 X 10(-4) s range) and to allow its lateral diffusion in the plane of the membrane fragments (observed by electron microscopy after freeze-etching or negative staining).
The DNA sequence of a clone from a cDNA library made from Xenopus laevis skin is described. This sequence represents the 3'-terminal end of an mRNA which codes for an epidermal cytokeratin polypeptide of mol. wt. 51 000 of the acidic (type I) subfamily as identified by hybridization-selection of mRNAs, followed by gel electrophoretic identification of the polypeptides synthesized by translation in vitro. The partial amino acid sequence of the amphibian cytokeratin shows strong similarity to type I cytoskeletal keratins from human (mol. wt. 50 000) and murine (mol. wt. 59 000) epidermis. In the non alpha-helical tail region the human and the non-mammalian (Xenopus) keratins are more similar to each other than to the murine protein, indicating that the former are equivalent cytokeratin polypeptides and belonging to a special subclass of type I keratin polypeptides devoid of glycine-rich regions in the carboxy-terminal portion. The evolutionary conservativity of the genes coding for cytokeratins is discussed.
P68 purified from d1331-infected cells has reduced kinase activity after dephosphorylation with bacterial alkaline phosphatase (BAP). The P68, purified from d1331-infected cells at 18 h post-infection utilizing the Mab-Sepharose, was pre-incubated for 30 min at 30°C in Buffer III containing: no BAP (lane B); 5 units BAP (lane C); and 10 units BAP (lane D). Lane A depicts the kinase activity of P68 which was not preincubated nor treated with BAP. Following BAP or mock treatment, the P68 was washed twice with Buffer H and twice with Buffer III. The kinase assay was then performed in Buffer m with 2 mM MgCl2, 1 mM MnCI2,
VAI RNA blocks the autophosphorylation of P68 present in the RSW of interferon-treated cells. Panel A: the RSW fraction from interferon-treated Daudi cells was prepared as described in Materials and methods. Reovirus dsRNA was then added at a concentration of 0.001 ,g/ml (lane B); 0.01 ,ug/ml (lane C); 0.1 tg/ml (lane D); 1.0 jig/ml (lane E); the reaction in lane A was performed in the absence of dsRNA. Protein kinase assays were done in Buffer III containing 2 mM MgCl2, 0.4 mM MnCl2, and [,y-32P]ATP. Samples were analyzed on a 10% SDS-polyacrylamide gel. Panel B: purified VAI RNA was added to the RSW at a concentration of 0.01 ,ug/ml (lane B); 0.1 tg/ml (lane C); 1.0 14g/mJ (lane D); 10.0 jAg/ml (lane E). The reaction shown in lane A contained no VAI RNA. 10.0 jg/ml purified 5S RNA was added to the kinase reaction shown in lane F. The kinase activity was assayed as described in Panel A. Panel C: the RSW salt wash was pre-incubated in Buffer III containing 2 mM MgCl2 and 0.4 mM MnCl2 for 15 min at 30°C with buffer alone (lanes A and B); 1.0 Agg/ml purified VAI RNA (lane C); 5.0 jg/ml VAI RNA (lane D); 10.0 14g/mn VAI RNA (lane E); and 10 jig/mn purified 5S RNA (lane F). Following the pre-incubation, reovirus dsRNA (0.01 /Ag/ml) was added to samples B-F and [-y32P]ATP to samples A-F for an additional 15 min incubation at 30°C. Samples were analyzed on a 10% polyacrylamide gel. Panel D: the P68 radioactive band in the autoradiogram shown in panel C was subjected to densitometer analysis: lanes A-F are identical to those described in panel C.  
We have investigated the interaction of VAI RNA with the interferon-induced, double-stranded (ds) RNA-activated protein kinase, P68, both of which regulate protein synthesis in adenovirus-infected cells. Previous work has shown that during infection by the VAI RNA-negative mutant, dl331, both viral and cellular protein synthesis are inhibited due to phosphorylation of the alpha-subunit of the eukaryotic initiation factor, eIF-2, by the P68 protein kinase. Utilizing monoclonal antibodies specific for P68, we demonstrated that the physical levels of P68 in dl331-infected, wild-type Ad2-infected and uninfected cells were all comparable suggesting that the elevated kinase activity detected during mutant infection was not due to increased P68 synthesis. To examine the basis of the increased activity of P68, the protein kinase was purified from infected-cell extracts using the monoclonal antibody. We found that P68 was heavily autophosphorylated during dl331 infection but not during wild-type or mock infection. The extent of autophosphorylation correlated with elevated P68 activity and the loss of the dsRNA requirements to phosphorylate the exogenous substrates, eIF-1 alpha and histones. We also analyzed VAI RNA function in vitro and present evidence that purified VAI RNA can block the autophosphorylation of P68 in the ribosomal salt wash fraction of interferon-treated cells. Finally we suggest VAI RNA functions through a direct interaction with the P68 protein kinase, since we demonstrated that VAI RNA forms a complex with P68 both in vitro and in vivo.
The chloroplast psbA gene from the green unicellular alga Chlamydomonas reinhardii has been localized, cloned and sequenced. This gene codes for the rapidly-labeled 32-kd protein of photosystem II, also identified as as herbicide-binding protein. Unlike psbA in higher plants which is found in the large single copy region of the chloroplast genome and is uninterrupted, psbA in C. reinhardii is located entirely within the inverted repeat, hence present in two identical copies per circular chloroplast genome, and contains four large introns. These introns range from 1.1 to 1.8 kb in size and fall into the category of Group I introns. Two of the introns contain open reading frames which are in-frame with the preceding exon sequences. We present the nucleotide sequence for the C. reinhardii psbA 5'-and 3' -flanking sequences, the coding region contained in five exons and the deduced amino acid sequence. The algal gene codes for a protein of 352 amino acid residues which is 95% homologous, excluding the last eight amino acid residues, with the higher plant protein.
Characterization and fractionation of RNA preparations from bovine muzzle epidermis as examined by one-dimensional gel electrophoresis of cell-free translation assays. (a) Coomassie Blue staining of a gel showing electrophoretically enriched desmosomal polypeptides D5 and D6 (arrowheads in lanes I and 3; lower mol. wt. bands represent some breakdown products from D5 and D6; for preparation see Mueller and Franke, 1983), endogenous unlabelled polypeptides present in the reticulocyte lysate used (lanes 2 and 4), blank (lane 5), and cytokeratin polypeptides I, III, IV, VI and VII from bovine muzzle epidermis (horizontal bars in lane 6). (b) Autoradiofluorography of the gel shown in (a), demonstrating the major products of translation in vitro (lanes 2' and 4') such as actin (A), cytokeratins VII, VI, 111, IV (the latter two are not labelled) and I as well as the two desmosomal polypeptides D6 and D5 (horizontal arrowheads). (c) Fractionation of RNA by gel electrophoresis on agarose presenting fractions examined by translation in vitro. Total unfractionated RNA (lane 1, cvtokeratin bands are labelled by a horizontal bar; A, actin) is compared with several fractions (lanes 2-8) including those containing the mRNA from desmosomal polypeptides (arrowheads) D6 (lane 5) and D5 (lanes 6 and 7). The horizontal arrow denotes a [35S]methionine-labelled endogenous protein present in the reticulocyte lysate. 
Isolated desmosomes from bovine epidermis contain two major polypeptides of mol. wts. 75 000 (D6) and 83 000 (D5) which, like the desmoplakins of mol. wt. greater than 200 000, are associated with the insoluble desmosomal plaque structure. We have characterized these two polypeptides and examined their significance by peptide map comparisons and translation of bovine epidermal mRNA in vitro. Polypeptide D5 is different from polypeptide D6 by its apparent mol. wt., its isoelectric pH (approximately 6.35, whereas D6 is a basic polypeptide isoelectric at pH approximately 8.5) and its peptide map. By all these criteria desmosomal polypeptides D5 and D6 are also different from cytokeratins, desmoplakins and the glycosylated desmosomal proteins. Both polypeptides are synthesized from different mRNAs separable by gel electrophoresis on agarose: mRNA coding for polypeptide D5 is approximately 3500 nucleotides long, that for D6 is significantly shorter (estimated to 3050 nucleotides), and both contain relatively large proportions of non-coding sequences. The translational products of these mRNAs co-migrate, on two-dimensional gel electrophoresis, with the specific polypeptides from bovine epidermis, indicating that they are genuine polypeptides and are not the result of considerable post-translational processing or modification of precursor molecules. The cell and tissue distribution of these two cytoskeletal proteins and possible functions are discussed.
Presequence protease PreP is a novel protease that degrades targeting peptides as well as other unstructured peptides in both mitochondria and chloroplasts. The first structure of PreP from Arabidopsis thaliana refined at 2.1 Angstroms resolution shows how the 995-residue polypeptide forms a unique proteolytic chamber of more than 10,000 Angstroms(3) in which the active site resides. Although there is no visible opening to the chamber, a peptide is bound to the active site. The closed conformation places previously unidentified residues from the C-terminal domain at the active site, separated by almost 800 residues in sequence to active site residues located in the N-terminal domain. Based on the structure, a novel mechanism for proteolysis is proposed involving hinge-bending motions that cause the protease to open and close in response to substrate binding. In support of this model, cysteine double mutants designed to keep the chamber covalently locked show no activity under oxidizing conditions. The manner in which substrates are processed inside the chamber is reminiscent of the proteasome; therefore, we refer to this protein as a peptidasome.
Cortical dysplasia in APP−/−APLP1−/−APLP2−/− triple knockout mice of various ages. (A, B) Frontal sections (cresyl violet staining) of a triple knockout mouse (T) at E18.5, exhibiting a prominent protrusion (P) of the right hemispheric cortical plate. The occurrence of ectopias was restricted to dorsal cortical areas as indicated by lines in (A). (B) Upon higher magnification (boxed area in A), it becomes apparent that ectopic neurons completely disrupt the subplate (SP) and cortical plate (CP); the cells have migrated into and beyond the MZ. No alterations were observed in the ventricular zone (not shown) (C, D) Depict low- and high-power images of a littermate control (C, genotype: APP−/−APLP1+/−APLP2−/−). (F) Example of an E17.5 triple knockout brain (T) showing two smaller cerebral protrusions (P). Arrowheads mark cells invading the subarachnoidal space (s). (E) Example of an E14.5 triple knockout exhibiting a small protrusion in comparison to a littermate control (C, genotype: APP−/−APLP1−/−APLP2+/−) depicted in (G). (H) Example of ectopia in a triple knockout embryo at E17.5. Note that a complete disruption of cortical layers is typically found within the center of dysplasias (B, H). In more lateral aspects (sections), the layering of deeper cortical structures appears much less affected (see, for example, F). Scale bars=500 μm (A, C), 100 μm (B, D, F, H), 50 μm (E, G).
Cortical dysplasia in APP À / À APLP1 À / À APLP2 À / À 
Immunohistochemical characterization of ectopias in APP À / À APLP1 À / À APLP2 À / À triple knockout mice at E18.5. ( A, B ) High-power 
Neuronal migration in the cerebral cortex is unaltered in triple mutants. (A–F) BrdU birthdating analysis. Pregnant mice were injected with BrdU either at E12.5 or at E15.5 and the distribution of BrdU þ neurons (green) was determined at E18.5 
Alterations in the MZ of triple knockout mice. ( A, B ) Appearance of the MZ in HE-stained sections of (A) littermate control and (B) triple knockout cortices at E16.5. Note the prominent reduction in the number of nuclei in the MZ of triple mutants. ( D, E ) Immunohistochemistry for reelin (red) revealed a consider- able reduction of CR cells in triple knockouts (E) as compared to controls (D) at E18.5. ( C ) The number of reelin-positive neurons within a 1200 m m wide MZ strip of the parietal cortex of E18.5 embryos was determined for both hemispheres on 8- m m-thick frozen frontal cortical sections. Values represent average cell counts 7 s.d. from n 1⁄4 8 stripes of four triple mutants (T, filled bar) and n 1⁄4 10 stripes of five littermate controls (C, open bar). * P o 0.05, Student’s t -test. ( F – K ) Frontal sections of E18.5 cortices from littermate control (F, I) and triple mutants (G, H, J, K) stained with antibodies against CSPG (F–H, red) or mDab1 (I–K, red). Cell nuclei were stained with DAPI (blue). The expression of CSPG and mDab1 within the MZ of triple knockouts (G, J) appeared unaffected in areas lacking protrusions. However, within protrusions (H, K), the normal pattern of expression of CSPG (H) and mDab1 (K) was interrupted and appeared disorganized. Scale bars 1⁄4 30 m m. Genotypes of littermate controls: APP À / À APLP1 À / À APLP2 þ / À (A, D), APP À / À APLP1 þ / þ APLP2 þ / þ (F), APP À / À APLP1 þ / þ APLP2 þ / À (I). 
The Alzheimer's disease beta-amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor-like proteins, termed APLP1 and APLP2. We previously documented that APLP2-/-APLP1-/- and APLP2-/-APP-/- mice die postnatally, while APLP1-/-APP-/- mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double-mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.
The nucleotide sequence of the Penicillium chrysogenum Oli13 acvA gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, which performs the first step in penicillin biosynthesis, has been determined. The acvA gene contains an open reading frame of 11,238 bp encoding a protein of 3746 amino acids with a predicted mol. wt of 421,073 dalton. Three domains within the protein of approximately 570 amino acids have between 38% and 43% identity with each other and share similarity with two antibiotic peptide synthetases from Bacillus brevis as well as two other enzymes capable of performing ATP-pyrophosphate exchange reactions. The acvA gene is located close to the pcbC gene encoding isopenicillin N synthetase, the enzyme for the second step of beta-lactam biosynthesis, and is transcribed in the opposite orientation to it. The intergenic region of 1107 bp from which the acvA and pcbC genes are divergently transcribed has also been sequenced.
The reaction of LEH with its natural substrate. Carbon atoms are numbered. 
Structure of LEH. ( A ) Ribbon drawing showing the dimer, with each subunit coloured going through the rainbow from red at the N-terminus to blue at the C-terminus. Some of the residues contributing to the dimer interface, as described in the text, are shown as ball-and-stick representations. The endogenous ligand is also shown (magenta) in both subunits. ( B ) Topology diagram of the LEH subunit, using the same rainbow scheme. Residues included in each secondary structural element are numbered; helix a 2 is irregular. Active-site residues are indicated by magenta stars. 
The active site. (A) Catalytic residues, showing their relationship to each other and supporting side-chains, as well as to the water molecule and endogenous ligand found in the LEH active site. Hydrogen-bonding interactions are shown by dotted lines. Colouring of the ribbon portions follows the rainbow scheme de®ned in Figure 2. The electron density of the endogenous ligand (modelled as heptanamide) in the ®nal 2F o ± Fc o map is contoured at a level of 1s. (B) Hydrogen-bonding interactions between active-site groups and the endogenous ligand. The ®gure shows Asp132 acting as the base and Asp101 as the acid. Where the donor±acceptor relationship is not clear from the available data, hydrogen bonds are indicated by doubleheaded arrows. (C) Complex with valpromide. The electron density of the ®nal 2F o ± Fc o map is contoured at a level of 1s.
Sequence alignments of LEH with proteins of unknown function. Sequence alignment was performed using hidden Markov models (Karplus et al ., 1998), with some small manual adjustments using the LEH structure as a guide. Every tenth residue in the LEH sequence is marked. Residues preceding a 1 are poorly conserved or missing in the other proteins, and so the alignments shown omit those segments. The roles that conserved residues play in LEH are indicated using the fol- lowing code: a, active site; c, core or other structure; d, dimer; s, surface. Members of the ®rst group of proteins, which appear to have both structural and functional relationships to LEH, are gi|2145793| (hypothetical protein B2235_F3_140 from Mycobacterium leprae ), gi|2624262| (hypothetical protein Rv2740 from Mycobacterium tuber- culosis ), gi:13424591| (hypothetical protein from Caulobacter crescen- tus ) and gi:17547308 (conserved hypothetical protein from the plant pathogen Ralstonia solanacearum ). Members of the second group, which are presumed to have a similar structure, but distinct function, are gi|11345638| (hypothetical protein VC1118 from Vibrio cholerae ) and gi|11350018| (hypothetical protein PA3856 from Pseudomonas aeruginosa ). A few regions may contain small shift errors, which will require new sequence/structural data to locate and correct. 
Mechanisms of epoxide hydrolases. ( A ) The mechanism of LEH indicated by the experimental results, using styrene oxide as an example. The catalytic water molecule is held in place and activated by hydrogen bonding to residues Asp132, Asn55 and Tyr53. The activated water molecule forces epoxide ring opening by nucleophilic attack at one of the ring carbons. At the same time, Asp101 activates the epoxide ring by donation of a proton to the epoxide oxygen (acid catalysis). Thus the formation of the diol from the epoxide proceeds in a single step by a push±pull mechanism. After this step, Asp132 should be in the protonated state and Asp101 should be charged, which can be rapidly reversed with the aid of Arg99 as a proton shuttle. The hydrogen bond donor/acceptor atoms for Tyr53 and Asp55 cannot be proved using current information, and only one of the two possibilities is drawn. ( B ) Reaction mechanism of a / b fold EHs. In these enzymes, two tyrosines position the epoxide oxygen by hydrogen bonding and activate the epoxide for nucleophilic attack by an aspartic acid residue. This ®rst step leads to the formation of an enzyme±substrate ester intermediate. Subsequent hydrolysis of the intermediate is achieved by a water molecule activated by a His-Asp/Glu charge relay system. The hydrolysis leads to product formation and reconstitution of the active enzyme. The tyrosines and charge relay system are only shown in the present scheme where they contribute to the mechanism. 
Epoxide hydrolases are essential for the processing of epoxide-containing compounds in detoxification or metabolism. The classic epoxide hydrolases have an alpha/beta hydrolase fold and act via a two-step reaction mechanism including an enzyme-substrate intermediate. We report here the structure of the limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis, solved using single-wavelength anomalous dispersion from a selenomethionine-substituted protein and refined at 1.2 A resolution. This enzyme represents a completely different structure and a novel one-step mechanism. The fold features a highly curved six-stranded mixed beta-sheet, with four alpha-helices packed onto it to create a deep pocket. Although most residues lining this pocket are hydrophobic, a cluster of polar groups, including an Asp-Arg-Asp triad, interact at its deepest point. Site-directed mutagenesis supports the conclusion that this is the active site. Further, a 1.7 A resolution structure shows the inhibitor valpromide bound at this position, with its polar atoms interacting directly with the residues of the triad. We suggest that several bacterial proteins of currently unknown function will share this structure and, in some cases, catalytic properties.
Electrophoretic analysis of active fractions obtained after cation and anion-exchange chromatographies. (A) Fractions from Sephadex G-25 (C), CM-cellulose (1) and PBE118 (2) were electrophoresed on SDS-polyacrylamide slab gels; proteins were stained with silver. (B) Fractions from Sephadex G-25 (C) and Q-Sepharose (3) were electrophoresed on polyacrylamide slab gels under native conditions; proteins were stained with Coomassie Blue; the position of acidic PR proteins in (C) is indicated. successive cationic and anionic exchange chromatographies. The enzymatic extract was first loaded onto a CM-cellulose column. The unbound protein fraction was dialysed against 20 mM Tris - HCl buffer, pH 7.8, and then applied to a Q-Sepharose column. 3210  
Three of the ten acidic ;pathogenesis-related' (PR) proteins known to accumulate in Nicotiana tabacum cv Samsun NN reacting hypersensitively to tobacco mosaic virus, namely -O, -N and -2, have been shown to have 1,3-beta-glucanase (EC activity. By using sera raised against each protein purified to homogeneity close serological relationships have been demonstrated between the three proteins. The same specific sera cross-reacted with a basic protein which is also a 1,3-beta-glucanase induced by virus infection and which can be considered as a new basic pathogenesis-related protein of tobacco. Protein PR-O and the basic 1,3-beta-glucanase display about the same specific enzymatic activity, i.e. 50-fold and 250-fold higher than specific activities of proteins PR-N and -2 respectively.
Endogenous glb sequences are targeted less ef®ciently by the silencing mechanism than corresponding transgene gn1 sequences. ( A ) Schematic presentation of the gn1 and glb mRNAs. Exons are indicated. The glb test regions eK and eL, and the corresponding gn1 test regions tK and tL, are represented with solid lines. The two K regions both have a length of 351 nt, and share 77% homology with a maximal stretch of 32 nt of uninterrupted homology. The two L regions both have a length of 297 nt, and share 83% homology with a maximal stretch of 36 nt of uninterrupted homology. Plasmids carrying the test regions between the STNV leader and trailer (Jacobs et al ., 1999) were linearized and in vitro transcribed to produce chimeric viral RNAs for delivery into protoplasts. ( B ) Northern blot analysis of total RNA extracted from protoplasts of hemizygous, expressing (He) and homozygous, silenced (Ho) T17 plants 20 h after delivery of TNV RNA and chimeric STNV RNA containing the test sequences shown in (A). 
Endogene-speci®c nucleotides contribute to sequence speci®city of the RNA degradation step of PTGS. ( A ) Upper part: position of the X, Y and Z test subregions within the K test region. Lower part: display of the homology between the glb , gn1 and arti®cial test sequences in the X, Y and Z subregions. The gray boxes show the `consensus sequence' between the transgenic, endogenous and arti®cial test sequences. Plasmids carrying the test regions between the STNV leader and trailer (Jacobs et al ., 1999) were linearized and in vitro transcribed to produce chimeric viral RNAs for delivery into protoplasts. ( B ) Features of the subregions X, Y and Z. The length of the X, Y and Z test sequences is shown, as well as the overall similarity between endogenous, transgenic and arti®cial sequences. For each subregion, the largest stretch of uninterrupted homology between the endogenous, transgenic and arti®cal sequences is shown. ( C ) Northern blot analysis of total RNA extracted from protoplasts of He and Ho T17 plants 20 h after delivery of TNV RNA and chimeric STNV RNA containing transgenic, endogenous and arti®cial test sequences as shown in (A). The 32 P-labeled RNA probe was complementary to the (+) strand of the STNV trailer. ( D ) Relative accumulation of chimeric STNV RNAs in protoplasts of hemizygous versus homozygous plants (He/Ho ratio). Bars represent the average of two (Z region) or three (X and Y region) independent experiments. CAT: STNV-CAT; the He/Ho ratio for the silencing-insensitive STNV CAT RNA is indicated by the horizontal dashed line. Letter codes above columns indicate statistical signi®cance of the differences between silencing susceptibilities within each region, as determined by repeated measures ANOVA. Columns marked a, b, c differ signi®cantly at 95% con®dence. The column marked a/b differs from those marked a and b at 88 and 80% con®dence, respectively. 
Glucanase silencing correlates with accumulation of small sense and antisense RNAs homologous to gn1 . ( A ) Nucleic acids from protoplasts and leaf tissue of hemizygous, expressing and homozygous, silenced T17 plants were enriched by PEG. Samples of 10 m g were separated on a 15% polyacrylamide gel and blotted to membranes. Filters were hybridized with 32 P-labeled RNA probes corresponding to the (+) 
Transgene- and endogene-speci®c small RNAs accumulate in protoplasts of silenced T17 plants. Nucleic acids from protoplasts and leaf tissue of hemizygous, expressing and homozygous, silenced T17 plants were enriched by PEG. Samples of 45 m g and in vitro synthesized control RNAs were separated on a 15% polyacrylamide gel, blotted onto membranes and hybridized with 32 P-labeled RNA probes corresponding to the (+) or (±) strand of transgenic and endogenous X region. The arrowheads indicate the position of the small RNA. Control RNAs: sense tX, sense eX: in vitro synthesized sense RNAs of ~70 nt corresponding to transgenic or endogenous region X; antisense tX, antisense eX: in vitro synthesized antisense RNAs of ~70 nt, corresponding to transgenic and endogenous region X. Probes for eX detection do not cross-hybridize to in vitro synthesized tX RNA and vice versa. 
Model explaining the activation of endogenous genes in PTGS. It assumes that transgene-speci®c (light gray) and `common' (black) siRNAs are produced from the inducing transgene. The small transgene-derived `common' antisense siRNAs anneal to homologous regions (black boxes) in the endogenous mRNA (a). The resulting partial dsRNA±RNA hybrids are recognized by an RNA-dependent RNA polymerase (RdRP) for elongation (b) or by the RNA-induced silencing complex (RISC) for direct degradation (dashed arrow). The dsRNA molecules resulting from RdRP activity are processed further by a Dicer-like enzyme, leading to the accumulation of secondary, endo- gene-derived siRNAs, the sequence of which can be endogene speci®c (dark gray) or common (black) (c). The endogene-speci®c secondary siRNAs can directly tag secondary targets such as endogene-speci®c regions in the endogenous mRNA (d), which subsequently become substrates for RISC-related degradation or a second round of RdRP- mediated dsRNA production and Dicer cleavage (dashed arrows). 
Post-transcriptional gene silencing (PTGS) is characterized by the accumulation of short interfering RNAs that are proposed to mediate sequence-specific degradation of cognate and secondary target mRNAs. In plants, it is unclear to what extent endogenous genes contribute to this process. Here, we address the role of the endogenous target genes in transgene-mediated PTGS of beta-1,3-glucanases in tobacco. We found that mRNA sequences of the endogenous glucanase glb gene with varying degrees of homology to the Nicotiana plumbaginifolia gn1 transgene are targeted by the silencing machinery, although less efficiently than corresponding transgene regions. Importantly, we show that endogene-specific nucleotides in the glb sequence provide specificity to the silencing process. Consistent with this finding, small sense and antisense 21- to 23-nucleotide RNAs homologous to the endogenous glb gene were detected. Combined, these data demonstrate that a co-suppressed endogenous glucan ase gene is involved in signal amplification and selection of homologous targets, and show that endogenous genes can actively participate in PTGS in plants. The findings are introduced as a further sophistication of the post-transciptional silencing model.
Analysis of,-1,3-glucanase activity in tobacco plants. (A) Direct assay for p3-1,3-glucanase activity on 7.5% IEF gel. T17 and NT correspond to total protein extracts from leaves of F3 T17 hemizygous and untransformed tobacco plants, respectively. (B) ,B-1,3-glucanase activity in total protein extracts from leaves of untransformed and F3 T17 hemizygous plants.  
Analysis of labelled run-off transcripts of homozygous and hemizygous T17 plants. Slot blots of linearized plasmids (gnl, nptII, ss and pGEM 2) were hybridized with labelled RNA transcripts from nuclei isolated from leaves of R3 homozygous and F3 hemizygous T17 plants as described in Materials and methods.  
Analysis of haploid plants. Haploid tobacco plants were generated as described in Materials and methods. (A) Chromosome number of homozygous (T17 Homo) and haploid (Hp Homo) tobacco plants. (B) RNA gel blot analysis of total leaf RNA isolated from independent haploid plants generated from anthers cultures of T17 homozygous (Hp Homo 1, 2 and 3) and T17 hemizygous (Hp He 1, 2 and 3) plants. RNA samples from R3 homozygous (T17 Homo) and F3 hemizygous (T17 He) plants are included as controls.  
A chimeric construct containing the Nicotiana plumbaginifolia beta-1,3-glucanase gn1 gene was introduced into Nicotiana tabacum SR1 to produce high levels of the enzyme constitutively. We determined that the GN1 protein represents a basic beta-1,3-glucanase isoform which accumulates into the vacuoles of the transgenic plants. Analysis of the progeny of the transgenic plant with the highest levels of gn1 expression revealed an unexpected phenomenon of gene suppression. Plants hemizygous for the T-DNA locus contained high levels of gn1 mRNA and exhibited a 14-fold higher beta-1,3-glucanase activity than untransformed plants. However, the expression of gn1 was completely suppressed in the homozygous plants: no corresponding mRNA or protein could be detected. This suppression mechanism occurs at a post-transcriptional level and is under developmental control. In addition, by generating haploid plants we found that this silencing phenomenon is not dependent on allelic interaction between T-DNA copies present at the same locus of homologous chromosomes, but rather is correlated with the transgene dose in the plant genome. We postulate that high doses of GN1 protein relative to the level(s) of other still unknown plant products could trigger the cellular processes directed to suppress gn1 expression.
SDS-PAGE of in vitro translation products obtained with 0.8 /%g of poly(A)+ RNA from non-induced (7 days, auxin + cytokinin medium) and induced (7 days, basal medium) tissues. Equal amounts (-2.5 x 105 c.p.m.) of 35S-labeled protein were used for immuno-precipitation with preimmune IgG or anti-glucanase IgG and were applied to the gels. Scale at left: mol. wt. (in kd) of protein standards. 
SDS-PAGE of in vitro translation products obtained with hybridselected RNA. Plasmid DNA from control (90) and glucanase (pGL43) clones was hybridized with total RNA from tissues incubated seven days on basal medium. Aliquots of the in vitro translation mixture and anti-glucanase immunoprecipitation were applied to the gels as indicated. Scale at left: mol. wt. (in kd) of protein standards. 
We describe the isolation of a cDNA clone of beta1,3-glucanase mRNA from Nicotiana tabacum L. cv. ;Havana 425' and its use to measure the kinetics of mRNA accumulation in cultured tobacco tissues treated with the plant hormones auxin and cytokinin. Northern blot analysis showed that the tissues contain a single 1.6 kb-sized beta1,3-glucanase mRNA. The levels of beta1,3-glucanase and beta1,3-glucanase mRNA increase by up to seven- and 20-fold, respectively, over a 7-day period in tissues subcultured on hormone-free medium and medium containing auxin or cytokinin added separately. Over the same interval of time, the content of both the enzyme and its mRNA remains at a constant low level in tissues subcultured on medium containing both auxin and cytokinin. The results show that auxin and cytokinin block beta1,3-glucanase production at the level of the mRNA.
Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.
Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell-cell and cell-matrix interactions. UDP-Gal:N-acetylglucosamine beta-1,4-galactosyltransferase (GalT) (EC is the enzyme which transfers galactose (Gal) to the terminal N-acetylglucosamine (GlcNAc) of complex-type N-glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell-cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT-deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT-deficient mice were born normally and were fertile, but they exhibited growth retardation and semi-lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.
Schematic representation of Iip31, Iip33 and hybrid proteins in which the cytoplasmic and membrane spanning domains have been partially or completely replaced by corresponding sequences from GT. The three domains depicted are the N-terminal (N) cytoplasmic domain, the membrane spanning domain, and the C-terminal (C) lumenal domain. The black boxes represent those parts of Iip31 which have been replaced by the corresponding sequences in GT. The expected sequence is numbered according to the published sequence for GT (Masri et al., 1988). We use the term membrane spanning domain to denote those amino acids thought to interact with the fatty acid chains of the lipids (underlined) and the one or two flanking amino acids thought to interact with the lipid head groups. The hatched box in Iip33 represents the additional 16 amino acids that cause retention in the ER.  
Immunofluorescence microscopy of HeLa cells transfected with (A) Iip33, (B) Iip3l, (C) GT1-12, (D) GT1-21, (E) GT1-47 and (F) GT1-58. Cells were fixed, permeabilized and incubated with antiserum reactive against the lumenal portion of Iip31. Bound antibody was visualized by a secondary antibody conjugated to Texas Red. Magnification at 800x.
Schematic representation of the hybrid protein, GT 1-47, after further truncation and substitution. Symbols as in Figure 1. 3570
Chimeric cDNAs were constructed so as to generate hybrid proteins in which different parts of the N-terminal domain of the human invariant chain were replaced by equivalent sequences from the trans Golgi resident enzyme, beta-1,4-galactosyltransferase. The cytoplasmic and membrane spanning domains of galactosyltransferase were found to be sufficient to retain all of the hybrid invariant chain in trans Golgi cisternae as judged by indirect immunofluorescence, treatment with brefeldin A and immuno-electron microscopy. As few as ten amino acids corresponding to the lumenal half of the membrane spanning domain of the Golgi enzyme sufficed to localize most of the hybrid invariant chain to the trans cisternae. A cytoplasmic domain was necessary for complete retention as assessed by flow cytofluorometry but could be provided either by galactosyltransferase or by invariant chain. This suggests that the cytoplasmic domain plays a role accessory to the membrane spanning domain, the latter mediating compartmental specificity.
Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.
The arylazide 1,4-dihydropyridine, [3H]azidopine, binds with high affinity to calcium channels in partially purified guinea-pig skeletal muscle transverse tubule membranes. Upon brief exposure to u.v. light, [3H]azidopine incorporates covalently into transverse tubule membrane proteins, as judged by SDS-PAGE. After alkylation of sulfhydryl groups with N-ethylmaleimide three specifically labelled bands of mol wts. 240 kd, 158 kd and 99 kd are always observed with fluorography after one-dimensional SDS-PAGE. Two other specific bands with mol. wts. of 52 kd and 55 kd, respectively, were sometimes observed. Two-dimensional SDS-PAGE (non-reduced but alkylated in the first dimension and reduced in the second dimension) revealed that the 240-kd band after reduction migrates with a mol. wt. of 99 kd. The 158-kd and 99-kd bands do not change in mobility. It is suggested that [3H]azidopine binds in such a way that the arylazide moiety of the ligand comes into contact with at least three calcium channel components: the A component of mol. wt. 240 kd, the B component of mol. wt. 158 kd and a C component of mol. wt. 99 kd. B and C are non-covalently bonded subunits of the channel, whereas A could be a heterodimer consisting of B and C, linked by disulfide bonds. Subunits of smaller mol. wt. may be also part of the ionic pore. Photolabelling of transverse tubule membranes after high energy irradiation with 10 MeV electrons supports this interpretation.
The three-dimensional structure of endo-1,4-beta-xylanase II (XYNII) from Trichoderma reesei has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 18.3% at 1.8 A resolution. The 190 amino acid length protein was found to exist as a single domain where the main chain folds to form two mostly antiparallel beta-sheets, which are packed against each other in parallel. The beta-sheet structure is twisted, forming a large cleft on one side of the molecule. The structure of XYNII resembles that of Bacillus 1,3-1,4-beta-glucanase. The cleft is an obvious suggestion for an active site, which has putative binding sites for at least four xylose residues. The catalytic residues are apparently the two glutamic acid residues (Glu86 and Glu177) in the middle of the cleft. One structure was determined at pH 5.0, corresponding to the pH optimum of XYNII. The second structure was determined at pH 6.5, where enzyme activity is reduced considerably. A clear structural change was observed, especially in the position of the side chain of Glu177. The observed conformational change is probably important for the mechanism of catalysis in XYNII.
CEACAM1 is a member of the carcinoembryonic antigen (CEA) family. Isoforms of murine CEACAM1 serve as receptors for mouse hepatitis virus (MHV), a murine coronavirus. Here we report the crystal structure of soluble murine sCEACAM1a[1,4], which is composed of two Ig-like domains and has MHV neutralizing activity. Its N-terminal domain has a uniquely folded CC' loop that encompasses key virus-binding residues. This is the first atomic structure of any member of the CEA family, and provides a prototypic architecture for functional exploration of CEA family members. We discuss the structural basis of virus receptor activities of murine CEACAM1 proteins, binding of Neisseria to human CEACAM1, and other homophilic and heterophilic interactions of CEA family members.
We examined the roles of inositol 1,4,5-trisphosphate (IP3) receptors (IP3R) in calcium signaling using DT40 B lymphocytes, and a variant lacking the three IP3R isoforms (IP3R-KO). In wild-type cells, B cell receptor (BCR) stimulation activates a cation entry route that exhibits significantly greater permeability to Ba2+ than does capacitative calcium entry. This cation entry is absent in IP3R-KO cells. Expression of the type-3 IP3R (IP3R-3) in the IP3R-KO cells rescued not only agonist-dependent release of intracellular Ca2+, but also Ba2+ influx following receptor stimulation. Similar results were obtained with an IP3R-3 mutant carrying a conservative point mutation in the selectivity filter region of the channel (D2477E); however, an IP3R-3 mutant in which this same aspartate was replaced by alanine (D2477A) failed to restore either BCR-induced Ca2+ release or receptor-dependent Ba2+ entry. These results suggest that in DT40 B lymphocytes, BCR stimulation activates a novel cation entry across the plasma membrane that depends upon, or is mediated by, fully functional IP3R.
Analysis with monoclonal antibodies. (A) Purified P4W protein was partially digested with Staphylococcus aureus V8 protease. The proteins were electrophoresed (5-12.5% SDS-PAGE) and immunodetected with 4Cl 1 (a), 1OA6 (b) and 18A10 (c) after the transfer of the proteins to a nitrocellulose sheet. The positions of mol. wt markers (in kd) are shown on the right. The triangle indicates the position of intact P4W protein. (B) Purified InsP3 receptor protein was electrophoresed (5% SDS-PAGE) and transferred to a nitrocellulose sheet. The blots were stained with amido black (a), or immunostained with 4C11 (b), 1OA6 (c) and 18A10 (d). 
indicates that InsP3 receptor activity was immunoprecipitated dose-dependently by 18A10 monoclonal 
P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.
Okadaic acid-sensitive phosphorylation of Ins(1,4,5)P 3 3-kinase A in rat brain cortical slices. Slices were preincubated with ortho-32 P for 2 h before incubation in the presence of various agents, i.e. carbachol (Cchol), okadaic acid (O.A.), KN-93, KN-62 and thapsigargin (TG). Slices were lysed in the presence of protease and phosphatase inhibitors and enzyme was immunoprecipitated using antirat brain Ins(1,4,5)P 3 3-kinase A antibodies. (A) A control where slices were incubated at 10 µM carbachol for 3 min before immunoprecipitation with preimmune serum (Pr.). The 53 kDa enzyme was detected by autoradiography (32 h exposure) following SDSPAGE. Carbachol was at 10 µM and okadaic acid at 100 nM. Okadaic acid was added for 30 min before stimulation. (B) KN-93 and KN-62 were added for a 30 min preincubation before slice stimulation with carbachol at 10 µM for 3 min. Thapsigargin was added for 3 min. 
CaM dependence of Ins(1,4,5)P 3 3-kinase activity in the presence of Ca 2 before and after phosphorylation in intact cells. (A) Coomassie Blue staining after SDS-PAGE of human brain Ins(1,4,5)P 3 3-kinase A (3 µg) purified on CaM-Sepharose from transfected CHO cells. (B) Enzyme was purified from CHO cells incubated in the presence (m) or absence (j) of 10 µM UTP for 30 s at 37°C. Enzymic activity was assayed at 5 µM Ins(1,4,5)P 3 and 10 µM free Ca 2 with increasing concentrations of CaM (0-1 µM). Results are means of triplicates SD. Fig. 3. Okadaic acid-sensitive phosphorylation of human Ins(1,4,5)P 3 3-kinase A in transfected CHO cells. Cells were preincubated with ortho-32 P for 2 h before incubation in the presence of various agents, 62 before receptor activation prevented 32 P incorporation 
Effect on enzyme activity and stoichiometry of phosphate 
Reverse phase HPLC of human brain Ins(1,4,5)P 3 3-kinase A 
D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] 3-kinase, the enzyme responsible for production of D-myo-inositol 1,3,4,5-tetrakisphosphate, was activated 3- to 5-fold in homogenates of rat brain cortical slices after incubation with carbachol. The effect was reproduced in response to UTP in Chinese hamster ovary (CHO) cells overexpressing Ins(1,4,5)P3 3-kinase A, the major isoform present in rat and human neuronal cells. In ortho-32P-labelled cells, the phosphorylated 53 kDa enzyme could be identified after receptor activation by immunoprecipitation. The time course of phosphorylation was very similar to that observed for carbachol (or UTP)-induced enzyme activation. Enzyme phosphorylation was prevented in the presence of okadaic acid. Calmodulin (CaM) kinase II inhibitors (i.e. KN-93 and KN-62) prevented phosphorylation of Ins(1,4,5)P3 3-kinase. Identification of the phosphorylation site in transfected CHO cells indicated that the phosphorylated residue was Thr311. This residue of the human brain sequence lies in an active site peptide segment corresponding to a CaM kinase II-mediated phosphorylation consensus site, i.e. Arg-Ala-Val-Thr. The same residue in Ins(1,4,5)P3 3-kinase A was also phosphorylated in vitro by CaM kinase II. Phosphorylation resulted in 8- to 10-fold enzyme activation and a 25-fold increase in sensitivity to the Ca2+:CaM complex. In this study, direct evidence is provided for a novel regulation mechanism for Ins(1,4,5)P3 3-kinase (isoform A) in vitro and in intact cells.
In the past few years, intracellular organelles, such as the endoplasmic reticulum, the nucleus and the mitochondria, have emerged as key determinants in the generation and transduction of Ca2+ signals of high spatio-temporal complexity. Little is known about the Golgi apparatus, despite the fact that Ca2+ within its lumen controls essential processes, such as protein processing and sorting. We report the direct monitoring of the [Ca2+] in the Golgi lumen ([Ca2+]Golgi) of living HeLa cells, using a specifically targeted Ca2+-sensitive photoprotein. With this probe, we show that, in resting cells, [Ca2+]Golgi is approximately 0.3 mM and that Ca2+ accumulation by the Golgi has properties distinct from those of the endoplasmic reticulum (as inferred by the sensitivity to specific inhibitors). Upon stimulation with histamine, an agonist coupled to the generation of inositol 1,4,5-trisphosphate (IP3), a large, rapid decrease in [Ca2+]Golgi is observed. The Golgi apparatus can thus be regarded as a bona fide IP3-sensitive intracellular Ca2+ store, a notion with major implications for the control of organelle function, as well as for the generation of local cytosolic Ca2+ signals.
Inositol 1,4,5-trisphosphate receptors (IP(3)Rs) are a family of intracellular Ca(2+) channels that exist as homo- or heterotetramers. In order to determine whether the N-terminal ligand-binding domain is in close physical proximity to the C-terminal pore domain, we prepared microsomal membranes from COS-7 cells expressing recombinant type I and type III IP(3)R isoforms. Trypsin digestion followed by cross-linking and co-immunoprecipitation of peptide fragments suggested an inter-subunit N- and C-terminal interaction in both homo- and heterotetramers. This observation was further supported by the ability of in vitro translated C-terminal peptides to interact specifically with an N-terminal fusion protein. Using a (45)Ca(2+) flux assay, we provide functional evidence that the ligand-binding domain of one subunit can gate the pore domain of an adjacent subunit. We conclude that common structural motifs are shared between the type I and type III IP(3)Rs and propose that the gating mechanism of IP(3)R Ca(2+) channels involves the association of the N-terminus of one subunit with the C-terminus of an adjacent subunit in both homo- and heterotetrameric complexes.
Stimulation of B-cell antigen receptor (BCR) induces a rapid increase in cytoplasmic free calcium due to its release from intracellular stores and influx from the extracellular environment. Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ligand-gated channels that release intracellular calcium stores in response to the second messenger, inositol 1,4,5-trisphosphate. Most hematopoietic cells, including B cells, express at least two of the three different types of IP3R. We demonstrate here that B cells in which a single type of IP3R has been deleted still mobilize calcium in response to BCR stimulation, whereas this calcium mobilization is abrogated in B cells lacking all three types of IP3R. Calcium mobilization by a transfected G protein-coupled receptor (muscarinic M1 receptor) was also abolished in only triple-deficient cells. Capacitative Ca2+ entry, stimulated by thapsigargin, remains unaffected by loss of all three types of IP3R. These data establish that IP3Rs are essential and functionally redundant mediators for both BCR- and muscarinic receptor-induced calcium mobilization, but not for thapsigargin-induced Ca2+ influx. We further show that the BCR-induced apoptosis is significantly inhibited by loss of all three types of IP3R, suggesting an important role for Ca2+ in the process of apoptosis.
(a) Immunofluorescence image of Purkinje neurons in bovine cerebellum. Conventional cryosections were stained with anti-IP3R antibody.  
(a) The same field as in Figure f after digitization. (b) Fourier spectrum of (a). The most prominent reflection spots are shown encircled, at a spacing of -16 nm and the next are shown with dotted circles at -19 nm. The sectioned specimen would include two adjacent membrane leaflets of cisternal stacks, giving rise to overlapping of two kinds of molecular arrays; one of which is the mirror image of the other. The stronger intensity of the first set of spots might be explained, at least partly, by the registered arrangement of the array to that direction, while the second set could split into weaker ones, since the lattice is not rectangular. (c) Selected spots used for reverse transform. These reflections were back-transformed to give the filtered image shown in (d). (e) The electron micrograph taken from the inset of Figure 5 of Takei et al. (1994) which was scanned and digitized for Fourier analysis. (f) Fourier spectrum of (e), whose most prominent peaks were extracted (g), and back-transformed to give the filtered image (h). The side length of the unit cell is 16.4 nm according to the authors' description. However, the actual ratio of spacing along two directions is -7:8 and not 1:1, as indicated here. It should also be noted that the two lines connecting each pair of reflection spots in (g) do not cross with a right angle but are inclined by -10°.  
(a) Arrays of receptor molecules whose distortion by the oblique view was corrected so that the membrane surface becomes almost normal to the line of vision. (b) Fourier spectrum of the field indicated in (a). (c) Selected spots used in the reverse transform. The distribution of reflection spots extends close to the 5 nm resolution limit. (d) Filtered image of (c). The molecular arrangement in the lattice and the tetrameric nature of each structural unit are evident. (e) Schematic diagram depicting the geometric arrangement of the molecules in the lattice. The dotted parallelogram indicates the elementary structural unit with the location of 2-fold rotation axes shown by pointed ellipses. The unit cell measures 14 nm (horizontal) and 16 nm (oblique), respectively. The ratio of these lengths is the same as found in the IP3R-overexpressing COS cell.  
(a) Triple tilted views of IP3R to give two pairs of stereo images. Each image was taken at 200 from its neighbour. Note the optimally shadowed particles which look elongated and have a spiral shape (arrowheads). (b) One stereo pair and three assorted images of RyaR molecules on SR vesicles. Images were taken using the same method as for IP3R, except for the use of mica flakes as the supporting material. Note the clear tetrameric structure and the crossed grooves on the surface of some of the subunits (arrowheads). The size of the total molecular assembly is almost twice that of IP3R. Scale bars represent 10 nm. See text for details.  
We used quick-freeze deep-etch replica electron microscopy to visualize the native structure of inositol-1,4,5-trisphosphate receptor (IP3R) in the cell. In the dendrites of Purkinje neurons of bovine cerebellum there were many vesicular organelles whose surfaces were covered with a two-dimensional crystalline array of molecules. Detailed examination of the cytoplasmic true surface of such vesicles in replica revealed that the structural unit, identified as IP3R by immunocytochemistry and subsequent Fourier analysis, is a square-shaped assembly and is aligned so that the side of the square is inclined by approximately 20 degrees from the row-line of the lattice. Comparison with the ryanodine receptor (RyaR), another intracellular Ca2+ channel on the endoplasmic reticulum, suggested that IP3R, unlike RyaR, has a very compact structure, potentially reflecting the crucial difference in the function of the cytoplasmic portion of the molecule.
Expression of DdPLC. Samples were taken from vegetatively growing AX3 and HD 1I (control cells) and HDIO (DdPLC gene disruption mutant). (A) Analysis of total RNA. The Northern blots were probed with DNA sequences encoding the conserved B-domain of PLC (left panel), the conserved A-domain of PLC (middle panel) and the tRNAGlU(UAA) (right panel). (B) Westem blot of proteins prepared from HDIO and HDI1 cells using DdPLC-specific antiserum. Numbers at the left indicate the migration position of molecular weight standards in kDa. 
The micro-organism Dictyostelium uses extracellular cAMP to induce chemotaxis and cell differentiation. Signals are transduced via surface receptors, which activate G proteins, to effector enzymes. The deduced protein sequence of Dictyostelium discoideum phosphatidylinositol-specific phospholipase C (PLC) shows strong homology with the mammalian PLC-delta isoforms. To study the role of PLC in Dictyostelium, a plc- mutant was constructed by disruption of the PLC gene. No basal or stimulated PLC activity could be measured during the whole developmental programme of the plc- cells. Loss of PLC activity did not result in a visible alteration of growth or development. Further analysis showed that developmental gene regulation, cAMP-mediated chemotaxis and activation of guanylyl and adenylyl cyclase were normal. Although the cells lack PLC activity, inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was present at only slightly lower concentrations compared with control cells. Mass analysis of inositol phosphates demonstrated the presence of a broad spectrum of inositol phosphates in Dictyostelium, which was unaltered in the plc- mutant. Cell labelling experiments with [3H]inositol indicated that [3H]Ins(1,4,5)P3 was formed in a different manner in the mutant than in control cells.
The inositol-1,4,5-triphosphate (InsP3) receptor consists of a homotetramer of highly conserved 313 kd subunits that contain multiple transmembrane regions in the C-terminal part of the protein. The receptor was expressed in COS cells and its domain structure was studied by mutagenesis. Deletion of the transmembrane regions from the receptor results in the synthesis of a soluble receptor protein that efficiently binds InsP3 but which instead of associating into homotetramers remains monomeric. This result suggests a role for the transmembrane regions in the association of the receptor subunits into tetramers but not in ligand binding. To localize the ligand binding site, further cDNAs encoding truncated receptor proteins were constructed. Assays of InsP3 binding to these truncated InsP3 receptors revealed that sequences in the N-terminal fourth of the InsP3 receptor are sufficient for ligand binding. Accordingly, each subunit of the InsP3 receptor homotetramer contains an independent ligand binding site that is located on the N-terminal ends of each subunit and is separated from the putative channel-forming transmembrane regions by greater than 1400 amino acids. Gel filtration experiments demonstrate a large conformational change of the receptor as a function of ligand binding, suggesting a mechanism by which ligand binding might cause channel opening.
Biochemical analysis of the puri®ed IP 3 Rs. (A) SDS±PAGE of the loading sample and the FPLC fractions around the IP 3 R peak. The gel was stained with Coomassie Blue. In lane 1, 5.0 mg of the loading sample was applied. Lanes 2±12 correspond to the fractions from the elution volume 6.0±15 ml. A 20 ml aliquot of each fraction was used for each lane. The protein concentration in fractions corresponding to lanes 4±6 was ~0.1 mg/ml. Fractions corresponding to these lanes were used in all other experiments. A control run of the high molecular weight standards showed that dextran 2000 came out at 7.8 ml and ferritin (437 kDa) at 14.5 ml, which correspond to lanes 4 and 12, respectively . (B) Immunoblotting of the puri®ed IP 3 Rs with speci®c antibodies against the type 1, 2 and 3 IP 3 Rs and an antibody against RyRs (both types 1 and 2). The positive control for the type 2 IP 3 R antibody was microsomes made from mouse cardiac muscle, and that for the type 3 IP 3 R antibody was made from renal cells (data not shown). The positive control (CTL) for the RyR antibody was the microsomes made from mouse skeletal muscle.  
Functional assay of the puri®ed IP 3 Rs. (A) [ 3 H]IP 3 binding from a competition assay. Shown here is the Scatchard plot, which was ®tted with an equation for the ratio of bound to free [ 3 H]IP 3 :  
Cryo-EM images of the IP 3 Rs and image processing. (A) A ®eld of IP 3 R particles, here visible as dark spots of ~150 A Ê diameter. The image was taken at 1.2 mm defocus, 42 000Q with a Tecnai 12 microscope operated at 120 kV. The electron dose for each exposure was ~10 e/A Ê 2 . (B) Projections of the converged model before CTF correction (left in each pair) compared with the corresponding class averages of the raw data (right in the pair). The orientation angles shown were sampled from the asymmetrical triangle. The two orientation angles (b and g) for each pair are shown next to them. The good match shows that the model is consistent with the data set.  
We report here the first three-dimensional structure of the type 1 inositol 1,4,5-trisphosphate receptor (IP(3)R). From cryo-electron microscopic images of purified receptors embedded in vitreous ice, a three-dimensional structure was determined by use of standard single particle reconstruction techniques. The structure is strikingly different from that of the ryanodine receptor at similar resolution despite molecular similarities between these two calcium release channels. The 24 A resolution structure of the IP(3)R takes the shape of an uneven dumbbell, and is approximately 170 A tall. Its larger end is bulky, with four arms protruding laterally by approximately 50 A and, in comparison with the receptor topology, probably corresponds to the cytoplasmic domain of the receptor. The lateral dimension at the height of the protruding arms is approximately 155 A. The smaller end, whose lateral dimension is approximately 100 A, has structural features indicative of the membrane-spanning domain. A central opening in this domain, which is occluded on the cytoplasmic half, outlines a pathway for calcium flow in the open state of the channel.
Inositol 1,4,5-trisphosphate (InsP3) was introduced into the cytoplasm of characean algae in two different ways: (i) by iontophoretic injection into cytoplasm-enriched fragments from Chara and (ii) by adding InsP3 to the permeabilization medium of locally permeabilized cells of Nitella. In both systems this operation induced a depolarization of the membrane potential, ranging from a few mV to sequences of action potentials. The effect of InsP3 on locally permeabilized Nitella cells was abolished when InsP3 was added together with 30 mM EGTA. When inositol 1,4-bisphosphate or myo-inositol were substituted for InsP3 in this system, there was no change in the membrane potential. On the other hand, increasing the free Ca2+ concentration in the permeabilization medium induced, in a similar fashion to InsP3, action potentials. Similarities between InsP3 and Ca2+ action were also observed upon injection into Chara fragments. Both injections increased an inward current. In the first few seconds after injection the current/voltage characteristics of the InsP3-induced current resembled those of the Ca2(+)-sensitive current. Subsequently, differences between the InsP3- and Ca2(+)-induced phenomena became apparent in that the InsP3-induced current continued to increase while the Ca2(+)-induced current declined, returning to the resting level. Our results suggest that these plant cells contain an InsP3 sensitive system that, under experimental conditions, is able to affect membrane transport via an increase in cytoplasmic free Ca2+.
Energized mitochondria increase the ability of InsP 3 to activate I CRAC in weak intracellular Ca 2+ buffer. ( A1 ) Time course of I CRAC to 
Effects of mitochondrial Ca 2+ buffering on the kinetics of I CRAC under conditions where SERCA pumps are active. The delay before I CRAC activates ( A1 ) and the time to peak ( A2 ) are plotted against InsP 3 concentration for cells with energized mitochondria. The time to peak was corrected for the delay. ( B1 ) The time course of I CRAC is shown for a cell dialysed with 30 m M InsP 3 in weak Ca 2+ buffer in the absence (open circles) and 
Moderate concentrations of the slow Ca 2+ chelator EGTA 
Cartoon summary of the role of mitochondria in activation of 
In eukaryotic cells, activation of cell surface receptors that couple to the phosphoinositide pathway evokes a biphasic increase in intracellular free Ca2+ concentration: an initial transient phase reflecting Ca2+ release from intracellular stores, followed by a plateau phase due to Ca2+ influx. A major component of this Ca2+ influx is store-dependent and often can be measured directly as the Ca2+ release-activated Ca2+ current (I(CRAC)). Under physiological conditions of weak intracellular Ca2+ buffering, respiring mitochondria play a central role in store-operated Ca2+ influx. They determine whether macroscopic I(CRAC) activates or not, to what extent and for how long. Here we describe an additional role for energized mitochondria: they reduce the amount of inositol 1,4,5-trisphosphate (InsP3) that is required to activate I(CRAC). By increasing the sensitivity of store-operated influx to InsP3, respiring mitochondria will determine whether modest levels of stimulation are capable of evoking Ca2+ entry or not. Mitochondrial Ca2+ buffering therefore increases the dynamic range of concentrations over which the InsP3 is able to function as the physiological messenger that triggers the activation of store-operated Ca2+ influx.
The therapeutic properties of lithium ions (Li+) are well known; however, the mechanism of their action remains unclear. To investigate this problem, we have isolated Li+-resistant mutants from Dictyostelium. Here, we describe the analysis of one of these mutants. This mutant lacks the Dictyostelium prolyl oligopeptidase gene (dpoA). We have examined the relationship between dpoA and the two major biological targets of lithium: glycogen synthase kinase 3 (GSK-3) and signal transduction via inositol (1,4,5) trisphosphate (IP3). We find no evidence for an interaction with GSK-3, but instead find that loss of dpoA causes an increased concentration of IP3. The same increase in IP3 is induced in wild-type cells by a prolyl oligopeptidase (POase) inhibitor. IP3 concentrations increase via an unconventional mechanism that involves enhanced dephosphorylation of inositol (1,3,4,5,6) pentakisphosphate. Loss of DpoA activity therefore counteracts the reduction in IP3 concentration caused by Li+ treatment. Abnormal POase activity is associated with both unipolar and bipolar depression; however, the function of POase in these conditions is unclear. Our results offer a novel mechanism that links POase activity to IP3 signalling and provides further clues for the action of Li+ in the treatment of depression.
Stereo view of the Ca backbone of the dimer of Rubisco. The colour scheme is as follows: Subunit 1: N-terminal domain: light blue, C-terminal domain: dark blue; Subunit 2: N-terminal domain: orange, C-terminal domain: red. 
Ca backbone of the N-terminal domain of one subunit of Rubisco. The strands of the mixed ,3-sheet are shown in yellow, the helices in pink and the connecting loops in blue. 
Ca backbone of the C-terminal domain of one subunit of Rubisco. The eight parallel (3-strands of the cx/(3 barrel are shown in yellow, the eight helices of the barrel are shown in pink. Connecting loops and additional secondary structure elements are shown in blue. 
Superposition of the a/,8 barrels of Rubisco (red) and glycolate oxidase (blue). The superposition is based on 158 equivalent atoms with an r.m.s. deviation of 2.7 A. The cofactor FMN bound to the active site in glycolate oxidase is shown in yellow. 
The three-dimensional structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum has been determined at 2.9 A resolution by X-ray crystallographic methods. The MIR-electron density map was substantially improved by two-fold non-crystallographic symmetry averaging. The polypeptide chains in the dimer were traced using a graphics display system with the help of the BONES option in FRODO. The dimer has approximate dimensions of 50 x 72 x 105 A. The enzyme subunit is a typical two-domain protein. The smaller, N-terminal domain consists of 137 amino acid residues and forms a central, mixed five-stranded beta-sheet with alpha-helices on both sides of the sheet. The larger C-terminal domain consists of 329 amino acid residues. This domain has an eight-stranded parallel alpha/beta barrel structure as found in triosephosphate isomerase and a number of other functionally non-related proteins. The active site in Rubisco determined by difference Fourier techniques and fitting of active site residues to the electron density map, is located at the carboxy-end of the beta-strands in the alpha/beta barrel of the C-terminal domain. There are few domain-domain interactions within the subunit. The interactions at the interface between the two subunits of the dimer are tight and extensive. There are tight contacts between the two C-terminal domains, which build up the core of the molecule. There are also interactions between the N-terminal domain of one subunit and the C-terminal domain of the second subunit, close to the active site.
Crystals of a tertiary complex of spinach ribulose-1,5-bisphosphate carboxylase/oxygenase with the activators Mg and CO(2) have been grown. These crystals diffract strongly to 1.6 A resolution. The spacegroup is C222(1) with unit cell dimensions a = 158.6 A, b = 158.6 A, c = 203.4 A. Additional local symmetry is apparent in the pattern of absences and the intensity distribution of the X-ray precession photographs. The photographs have been interpreted in terms of a molecule (consisting of eight large and eight small subunits, L(8)S(8)) with 222 symmetry and a molecular centre shifted 2 A in the x direction from the origin of the unit cell. The asymmetric unit contains half the L(8)S(8) molecule. The intensity distribution suggests that the molecular symmetry does not deviate far from 422. These crystals are compared with other crystalline forms of the enzyme and the implications of these results are discussed.
In vitro mutagenic techniques have generated an asp-->glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C-->A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.
Schematic picture of the subunit of Rubisco from R. rubrum. Helices are shown as cylinders and ,B-strands as arrows. The nomenclature of the secondary structural elements is included. The small arrows indicate the points at which the polypeptide chain was truncated in the various mutants. 440 450 Spinach...L A R E G N T I I R E A T K W S aCl 
Electrophoresis of purified Rubisco in the absence (A) or presence (B) of SDS. Lanes 1 and 5: wild-type Rubisco; Lanes 2, 3 and 4: mutant protein excised at position 458, 449 or 441, respectively. Lane 6: molecular weight markers. A polyacrylamide gradient gel (5-15%) and a homogeneous gel (10% in acrylamide) were used for non-denaturing and SDS electrophoresis, respectively. Proteins were stained in 0. 1 % Coomassie blue. 
Immunodetection of Rubisco from cell lysates of E. coli after electrophoresis as in Figure 3. E. coli cells contain wild-type Rubisco (lane 1) or mutant P441 before (lane 2) and after L424 to N424 substitution (lane 3). 
Truncations of the subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Rhodospirillum rubrum were generated by site-directed mutagenesis to examine the role of the C-terminal tail section. Removal of the last and the penultimate alpha-helices in the tail section changes the quaternary structure of the protein. Electrophoretic and electron microscope analysis revealed that the truncated subunits assemble into an octamer, whereas the wild-type enzyme has a dimeric structure. The octomerization of the mutant protein is due to a hydrophobic patch exposed to the solvent by truncation of the subunit. The mutant protein thus consists of four dimers, bound end-to-end by hydrophobic interactions. Insertion of a polar amino acid in the hydrophobic patch by a L424 to N424 substitution restores the familiar dimeric structure. Truncation of the subunit is associated with a considerable decrease in catalytic activity. The mutants undergo carbamylation but bind the reaction intermediate analog, 2-carboxy arabinitol-1,5-bisphosphate, poorly. This indicates that loss of activity in the mutant is due to weakened substrate binding. These findings suggest that the mutations in the tail section of the subunit are transmitted to the active site, although the C-terminal region is far from the active site. On the basis of the crystal structure of Rubisco, we propose a model for how the truncations of the enzyme subunit induce conformational changes in one of the two phosphate binding sites.
Transcripts of three distinct ribulose-1,5-bisphosphate carboxylase (RuBPC) small subunit (SS) genes account for approximately 90% of the mRNA for this protein in maize leaves. Transcripts of two of them constitute >80% of the SS mRNA in 24-h greening maize leaves. The third gene contribute approximately 10%. Transcripts of all three nuclear-encoded SS genes are detectable in bundle sheath (BSC) and mesophyll cells (MC) of etiolated maize leaves. The level of mRNA for each gene is different in etioplasts of MC but all drop during photoregulated development of chloroplasts in MC and follow a pattern of transitory rise and fall in BSC. The amounts of LS and SS proteins continue to increase steadily well after the mRNA levels reach their peaks in BSC. The molar ratio of mRNA for chloroplast-encoded RuBPC large subunit (LS) to the nuclear genome encoded SS is about 10:1 although LS and SS proteins are present in about equimolar amounts.
In photosynthetic eukaryotes, the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is composed of eight large and eight small subunits. Chloroplast-coded large subunits are found in association with chaperonins (binding proteins) of 60-61 kd to form a high mol. wt pre-assembly complex (B-complex). We have isolated a heterotrophic, maternally-inherited mutant from Nicotiana tabacum var. Xanthi which accumulates the B-complex but contains no Rubisco holoenzyme. The B-complex of the mutant dissociates in the presence of ATP, as does that of the wild-type. Processing of the nuclear-coded small subunit takes place in the mutant and neither large nor small subunits accumulate. The large subunit gene from mutant and wild-type plants was cloned and sequenced. A single nucleotide difference was found between them predicting an amino acid change of serine to phenylalanine at position 112 in the mutant. Based on the resolved structure of N.tabacum Rubisco, it is argued that the alteration at position 112 prevents holoenzyme assembly by interfering with large subunit assembly.
The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is synthesized when cells of the yeast Saccharomyces cerevisiae are grown on a non-fermentable carbon source. After shifting the cells to glucose-containing medium, in a process called catabolite degradation, FBPase is selectively and rapidly broken down. We have isolated gid mutants, which are defective in this glucose-induced degradation process. When complementing the defect in catabolite degradation of FBPase in gid3-1 mutant cells with a yeast genomic library, we identified the GID3 gene and found it to be identical to UBC8 encoding the ubiquitin-conjugating enzyme Ubc8p. The in vivo function of Ubc8p (Gid3p) has remained a mystery so far. Here we demonstrate the involvement of Ubc8p in the glucose-induced ubiquitylation of FBPase as a prerequisite for catabolite degradation of the enzyme via the proteasome. Like FBPase, Ubc8p is found in the cytoplasmic fraction of the cell. We demonstrate cytoplasmic degradation of FBPase.
The role of glucose trimming in the endoplasmic reticulum of Saccharomyces cerevisiae was investigated using glucosidase inhibitors and mutant strains devoid of glucosidases I and II. These glucosidases are responsible for removing glucose residues from the N-linked core oligosaccharides attached to newly synthesized polypeptide chains. In mammalian cells they participate together with calnexin, calreticulin and UDP-glucose:glycoprotein glucosyltransferase in the folding and quality control of newly synthesized glycoproteins. In S.cerevisiae, glucosidase II is encoded by the GLS2 gene, and glucosidase I, as suggested here, by the CWH41 gene. Using castanospermine (an alpha-glucosidase inhibitor) and yeast strains defective in glucosidase I, glucosidase II and BiP/Kar2p, it was demonstrated that cell wall synthesis depends on the two glucosidases and BiP/Kar2p. In double mutants with defects in both BiP/Kar2p and either of the glucosidases the phenotype was particularly clear: synthesis of 1,6-beta-glucan_a cell wall component_was reduced; the cell wall displayed abnormal morphology; the cells aggregated; and their growth was severely inhibited. No defects in protein folding or secretion could be detected. We concluded that glucose trimming in S.cerevisiae is necessary for proper cell wall synthesis, and that the glucosidases function synergistically with BiP/Kar2p in this process.
Anp1p, Van1p and Mnn9p constitute a family of membrane proteins required for proper Golgi function in Saccharomyces cerevisiae. We demonstrate that these proteins colocalize within the cis Golgi, and that they are physically associated in two distinct complexes, both of which contain Mnn9p. Furthermore, we identify two new proteins in the Anp1p-Mnn9p-containing complex which have homology to known glycosyltransferases. Both protein complexes have alpha-1, 6-mannosyltransferase activity, forming a series of poly-mannose structures. These reaction products also contain some alpha-1, 2-linked mannose residues. Our data suggest that these two multi-protein complexes are responsible for the synthesis and initial branching of the long alpha-1,6-linked backbone of the hypermannose structure attached to many yeast glycoproteins.
The immune response to beta-(1,6)-galactan in the BALB/c mouse has been well characterized and includes the amino acid sequence determination of 13 monoclonal antibodies. The genetic potential encoding the VH regions of these antibodies has been determined by isolation and sequencing of homologous germline genes. The germline repertoire encoding these proteins was found to consist of two closely related genes. One of these directly encodes the VH segments of seven Gal-binding proteins, and the second directly encodes one additional protein sequence. Sequence variations found in the VH regions of five other Gal-binding proteins can be explained by somatic mutations leading to single base substitutions in the more frequently used gene. Since four of the hybridoma proteins exhibiting somatic mutations are of the IgM class, these results indicate that somatic mutation, in this system, is not associated with class switching and can apparently be initiated early in B-cell development. The two Gal genes are the only members of a very restricted multigene family and probably result from a gene duplication estimated to occur 1.4-2.8 million years ago. Three other genes hybridizing at moderate stringency to a VHGal probe were also sequenced and were found to be members of two additional VHIII families. Studies of the silent to replacement substitution ratios of these and other VH genes indicate that the number of silent substitutions found in immunoglobulin VH genes is lower than expected when compared with proteins such as preproinsulin and globin. Analysis of base composition reflected in these sequences indicates a marked increase of A-T% in the first and second codon positions of complementarity determining regions (CDR) which may be important in facilitating point mutations.
The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.
Structure of oxidized FBPase. (A) The wild-type form I FBPase tetramer. The insertion (residues 153-173) is in red, the disulfide bridge in green, strands β1 and β2 (residues 99-118) in yellow. Dashed lines indicate regions of weak electron density. The dimers are in light pink/yellow and in shades of blue. (B) The disulfide bridge between Cys153 and Cys173. |F o |-|F c | electron density of wild-type form I FBPase with the disulfide bridge omitted from the calculation, shown at 5σ cutoff. Residues 152 and 174-177 from the insertion are shown as main chain trace. Cys178 is located on the side of the helix that faces the core of the enzyme.
Comparison of oxidized pea FBPase to pig kidney and spinach FBPases. (A) Oxidized form I pea FBPase. (B) Chloroplastic spinach FBPase (PDB entry code 1SPI) (Villeret et al., 1995). (C) R form gluconeogenic pig kidney FBPase in complex with F6P, P i and Zn 2 (PDB entry code 1CNQ) (Choe et al., 1998). (D) Close-up view of the cation binding site with pig kidney FBPase shown in blue, cations in red and oxidized pea FBPase in yellow. Orientation and colour coding in (A), (B) and (C) are as in Figure 1A. The location of the active site in pea and spinach FBPases is indicated by a model of F6P, P i and Zn 2 shown as dashed lines. The 70's loop in pig kidney FBPase is in blue. The corresponding loop is disordered in pea and spinach FBPases. The loop in pig kidney FBPase that corresponds to the chloroplastic insertion is in red. The binding site for AMP in pig kidney FBPase is indicated by one of its ligands, Lys112. The close-up view in (D) shows that the interaction of the 70's loop and the loop between strands β1 and β2 with the cations in pig FBPase, is prevented by the inwards movement of strands β1and β2 in oxidized pea FBPase. This movement places Val109 near the location of the cation binding site and removes Glu105, which corresponds to Glu97 in pig kidney FBPase, from the active site.
The insertion in wild-type form I (dark grey) and in the Cys173Ser mutant (light grey). Residues 155 – 168 are weakly de fi ned in the electron density of form I (dashed lines) and cannot be traced in the C173S mutant. 
The quaternary structures of wild-type form I (left) and wild-type form II (right) FBPases. The reference dimer (in grey) has the same orientation in both forms. The 2-fold axis relating monomers of the 
Sunlight provides the energy source for the assimilation of carbon dioxide by photosynthesis, but it also provides regulatory signals that switch on specific sets of enzymes involved in the alternation of light and dark metabolisms in chloroplasts. Capture of photons by chlorophyll pigments triggers redox cascades that ultimately activate target enzymes via the reduction of regulatory disulfide bridges by thioredoxins. Here we report the structure of the oxidized, low-activity form of chloroplastic fructose-1, 6-bisphosphate phosphatase (FBPase), one of the four enzymes of the Calvin cycle whose activity is redox-regulated by light. The regulation is of allosteric nature, with a disulfide bridge promoting the disruption of the catalytic site across a distance of 20 A. Unexpectedly, regulation of plant FBPases by thiol-disulfide interchange differs in every respect from the regulation of mammalian gluconeogenic FBPases by AMP. We also report a second crystal form of oxidized FBPase whose tetrameric structure departs markedly from D(2) symmetry, a rare event in oligomeric structures, and the structure of a constitutively active mutant that is unable to form the regulatory disulfide bridge. Altogether, these structures provide a structural basis for redox regulation in the chloroplast.
Top-cited authors
Junying Yuan
  • Harvard Medical School
Guido Kroemer
  • Paris Descartes, CPSC
Guy Las
  • Ben-Gurion University of the Negev
Jakob Wikstrom
  • Karolinska Institutet
Gilad Twig
  • Sheba Medical Center