The EMBO Journal

Published by Wiley

Online ISSN: 1460-2075


Print ISSN: 0261-4189


Figure 1 of 1
Chlamydomonas reinhardii gene for the 32 000 mol. wt. protein of Photosystem II contains four large introns and is located entirely within the chloroplast inverted repeat
  • Article
  • Full-text available

January 1985


96 Reads

Jeanne M. Erickson


Michèle Rahire


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.

Fig. 4. 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,
Fig. 5. 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.  
Adenovirus VAI RNA complexes with the 68 000 Mr protein kinase to regulate its autophosphorylation and activity

April 1987


44 Reads

M G Katze


D DeCorato


B Safer




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.

Fig. 3. 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. 
Figure 2 of 2
Significance of two desmosome plaque-associated polypeptides of molecular weights 75 000 and 83 000.

February 1983


47 Reads

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.

Fig. 7. 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. 
Figure 2 of 3
Figure 3 of 3
Synaptobrevin: An integral membrane protein of 18 000 daltons present in small synaptic vesicles of rat brain

March 1989


72 Reads

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.

The closed structure of presequence protease PreP forms a unique 10 000 Å3 chamber for proteolysis

June 2006


146 Reads

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.

The human ubiquitin carrier protein E2(Mr =17 000) is homologous to the yeast DNA repair gene RAD6

June 1990


37 Reads

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.

Fig. 3. 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 receptor in Torpeda marmorata membrane fragments studied with a spin-labelled alpha-toxin: importance of the 43 000 protein(s)

February 1982


53 Reads

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).

Amino acid sequence of the carboxy-terminal part of an acidic type I cytokeratin of molecular weight 51 000 from Xenopus laevis epidermis as predicted from the cDNA sequence

July 1984


27 Reads

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.

Fig. 3. 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. 
Figure 2 of 3
Figure 3 of 3
Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts

February 1985


69 Reads

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.

Fig. 6. 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  
Phosphorylation of an acidic mol. wt. 80 000 cellular protein in a cell-free system and intact Swiss 3T3 cells: a specific marker of protein kinase C activity

February 1986


28 Reads

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.

Figure 1 of 1
Targeted disruption of the Mr 46 000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins

January 1994


48 Reads

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.

Figure 1: 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).
Figure 1 Cortical dysplasia in APP À / À APLP1 À / À APLP2 À / À 
Figure 2 Immunohistochemical characterization of ectopias in APP À / À APLP1 À / À APLP2 À / À triple knockout mice at E18.5. ( A, B ) High-power 
Figure 3 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 
Figure 4 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). 
O4-04-07 Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP-family members

November 2004


342 Reads

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 multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421 073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases

October 1990


30 Reads

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.

Fig. 1. The reaction of LEH with its natural substrate. Carbon atoms are numbered. 
Fig. 2. 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. 
Fig. 3. 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.
Fig. 4. 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. 
Fig. 5. 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. 
Structure of Rhodococcus erythropolis limonene-1,2-epoxide hydrolase reveals a novel active site

July 2003


566 Reads

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.

Fig. 3. 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  
Biological function of `pathogenesis-related' proteins: four PR proteins of tobacco have 1,3-β-glucanase activity

December 1987


171 Reads

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.

Fig. 1. 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. 
Fig. 2. 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. 
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Hormonal regulation of β-1,3-glucanase messenger RNA levels in cultured tobacco tissues

August 1985


88 Reads

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.

Fig. 1. 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). 
Fig. 2. 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. 
Fig. 3. 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 (+) 
Fig. 4. 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. 
Fig. 5. 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). 
An active role for endogenous β-1,3-glucanase genes in transgene-mediated co-suppression in tobacco

December 2002


65 Reads

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.

Fig. 5. 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.  
Fig. 7. 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.  
Fig. 8. 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.  
Suppression of β-1,3-glucanase transgene expression in homozygous plants

August 1992


95 Reads

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.

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A

May 1987


36 Reads

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+.

Growth retardation and early death of β-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells

May 1997


240 Reads

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.

Crystal structure of murine sCEACAM1a[1,4]: A coronavirus receptor in the CEA family

June 2002


197 Reads

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.

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Subunit composition of skeletal muscle transverse tubule calcium channels evaluated with the 1,4-dihydropyridine photoaffinity probe, [3H]azidopine

September 1985


31 Reads

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.

Fig. 1. 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.  
Fig. 2. 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.
Fig. 4. Schematic representation of the hybrid protein, GT 1-47, after further truncation and substitution. Symbols as in Figure 1. 3570
The membrane spanning domain of β-1,4-galactosyltransferase specifies trans Golgi localization

January 1992


75 Reads

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.

A plasma membrane-bound putative endo-1,4-??-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis

November 1998


252 Reads

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.

Three-dimensional structure of endo-1,4-??-xylanase II from Trichoderma reesei: Two conformational states in the active site

July 1994


72 Reads

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.

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