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ABSTRACT: The p150-Spir protein, which was discovered as a phosphorylation target of the Jun N-terminal kinase, is an essential regulator of the polarization of the Drosophila oocyte. Spir proteins are highly conserved between species and belong to the family of Wiskott-Aldrich homology region 2 (WH2) proteins involved in actin organization. The C-terminal region of Spir encodes a zinc finger structure highly homologous to FYVE motifs. A region with high homology between the Spir family proteins is located adjacent (N-terminal) to the modified FYVE domain and is designated as "Spir-box." The Spir-box has sequence similarity to a region of rabphilin-3A, which mediates interaction with the small GTPase Rab3A. Coexpression of p150-Spir and green fluorescent protein-tagged Rab GTPases in NIH 3T3 cells revealed that the Spir protein colocalized specifically with the Rab11 GTPase, which is localized at the trans-Golgi network (TGN), post-Golgi vesicles, and the recycling endosome. The distinct Spir localization pattern was dependent on the integrity of the modified FYVE finger motif and the Spir-box. Overexpression of a mouse Spir-1 dominant interfering mutant strongly inhibited the transport of the vesicular stomatitis virus G (VSV G) protein to the plasma membrane. The viral protein was arrested in membrane structures, largely colocalizing with the TGN marker TGN46. Our findings that the Spir actin organizer is targeted to intracellular membrane structures by its modified FYVE zinc finger and is involved in vesicle transport processes provide a novel link between actin organization and intracellular transport.
Current Biology 01/2002; 11(24):1963-8. · 9.65 Impact Factor
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ABSTRACT: Taking each coding sequence from the human genome in turn and identifying the subcellular localization of the corresponding protein would be a significant contribution to understanding the function of each of these genes and to deciphering functional networks. This article highlights current approaches aimed at achieving this goal.
Genome biology 02/2001; 2(9):REVIEWS1024. · 6.63 Impact Factor
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ABSTRACT: As a first step towards a more comprehensive functional characterization of cDNAs than bioinformatic analysis, which can only make functional predictions for about half of the cDNAs sequenced, we have developed and tested a strategy that allows their systematic and fast subcellular localization. We have used a novel cloning technology to rapidly generate N- and C-terminal green fluorescent protein fusions of cDNAs to examine the intracellular localizations of > 100 expressed fusion proteins in living cells. The entire analysis is suitable for automation, which will be important for scaling up throughput. For > 80% of these new proteins a clear intracellular localization to known structures or organelles could be determined. For the cDNAs where bioinformatic analyses were able to predict possible identities, the localization was able to support these predictions in 75% of cases. For those cDNAs where no homologies could be predicted, the localization data represent the first information.
EMBO Reports 09/2000; 1(3):287-92. · 7.36 Impact Factor
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ABSTRACT: The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway. Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules. Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER. Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited. In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes. Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins. These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent. The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.
Nature Cell Biology 12/1999; 1(7):423-30. · 19.49 Impact Factor
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ABSTRACT: Cytotoxic proteins such as ricin A chain (RTA) have target substrates in the cytosol and therefore have to reach this cellular compartment in order to act. RTA is thought to translocate into the cytosol from the lumen of the endoplasmic reticulum (ER), although how it traverses the ER membrane has not been established. Using yeast mutants defective in various aspects of the ER-associated protein degradation (ERAD) pathway, we show that RTA introduced into the yeast ER subverts this pathway to enter the cytosol via the Sec61p translocon. A significant proportion of the exported RTA avoided proteasomal degradation. These data are consistent with the contention that the RTA component from ricin endocytosed by mammalian cells may likewise exploit ERAD to translocate into the cytosol.
FEBS Letters 11/1999; 459(1):80-4. · 3.54 Impact Factor
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ABSTRACT: To investigate the role of the KDEL receptor in the retrieval of protein toxins to the mammalian cell endoplasmic reticulum (ER), lysozyme variants containing AARL or KDEL C-terminal tags, or the human KDEL receptor, have been expressed in toxin-treated COS 7 and HeLa cells. Expression of the lysozyme variants and the KDEL receptor was confirmed by immunofluorescence. When such cells were challenged with diphtheria toxin (DT) or Escherichia coli Shiga-like toxin 1 (SLT-1), there was no observable difference in their sensitivities as compared to cells which did not express these exogenous proteins. By contrast, the cytotoxicity of Pseudomonas exotoxin A (PE) is reduced by expressing lysozyme-KDEL, which causes a redistribution of the KDEL receptor from the Golgi complex to the ER, and cells are sensitised to this toxin when they express additional KDEL receptors. These data suggest that, in contrast to SLT-1, PE can exploit the KDEL receptor in order to reach the ER lumen where it is believed that membrane transfer to the cytosol occurs. This contention was confirmed by microinjecting into Vero cells antibodies raised against the cytoplasmically exposed tail of the KDEL receptor. Immunofluorescence confirmed that these antibodies prevented the retrograde transport of the KDEL receptor from the Golgi complex to the ER, and this in turn reduced the cytotoxicity of PE, but not that of SLT-1, to these cells.
Journal of Cell Science 03/1999; 112 ( Pt 4):467-75. · 6.11 Impact Factor
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ABSTRACT: Diphtheria toxin is believed to enter sensitive mammalian cells via receptor-mediated endocytosis from clathrin-coated pits, while ricin can enter via both clathrin-dependent and clathrin-independent endocytosis. The present study has confirmed this by determining the toxin sensitivity of COS-7y cells which were transiently overexpressing a trans dominant negative mutant of dynamin, a GTPase required for the budding of clathrin-coated vesicles from the plasma membrane. Cells overexpressing wild-type dynamin showed normal receptor-mediated endocytosis of transferrin and remained sensitive to both diphtheria toxin and ricin. Cells overexpressing a mutant dynamin defective in GTP binding and hydrolysis were unable to endocytose transferrin and were protected against diphtheria toxin, but they remained completely sensitive to ricin intoxication. Treating non-transfected cells or cells overexpressing mutant dynamin with nystatin caused a redistribution of the caveolae membrane marker protein VIP21-caveolin from the cell surface to intracellular locations, but did not affect their sensitivity to ricin. The redistribution of caveolin seen after nystatin treatment may reflect the disappearance of caveolae. If this is the case, caveolae are not responsible for the endocytosis of ricin. An alternative clathrin-independent route may operate for ricin, since cellular uptake, intracellular transport, and translocation into the cytosol remain unaffected when clathrin-dependent endocytosis is effectively blocked.
Experimental Cell Research 03/1998; 239(2):293-300. · 3.58 Impact Factor
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ABSTRACT: During the intoxication of mammalian cells by ricin, the catalytically active A chain must cross the membrane of an intracellular compartment in order to reach its ribosomal substrates in the cytosol. The actual site of ricin A chain translocation is unclear, and conflicting views hold that it enters the cytosol from endosomes or from an early compartment of the secretory pathway, possibly the lumen of the endoplasmic reticulum. Here we show that treating cells with brefeldin A, or transiently overexpressing mutant GTPases known to inhibit biochemical complexes mediating anterograde and retrograde transport between the endoplasmic reticulum and the Golgi complex, protected cells from intoxication by free ricin A chain. These data indicate that ricin A chain, either free or as part of intact ricin, reaches an early compartment of the secretory pathway before translocation into the cytosol occurs.
Experimental Cell Research 01/1997; 229(2):447-51. · 3.58 Impact Factor
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ABSTRACT: Ricin A chain (RTA) mutants which had been modified by the addition of three lysine residues, three lysines and an alanine, or six histidine residues at the carboxyl terminus were expressed in Escherichia coli. The recombinant proteins were purified to homogeneity by ion-exchange chromatography on CM-Sepharose CL-6B. The 28S ribosomal RNA N-glycosidase activities of the three RTA mutants were indistinguishable from each other and from the activity of wild-type recombinant RTA. The RTA mutants were not impaired, compared with wild-type RTA, in their ability to reassociate with ricin B chain to form ricin holotoxin. Holotoxins containing mutant RTAs were as readily dissociated into subunits under reducing conditions as native holotoxin, and the RTA mutants were indistinguishable from wild-type RTA in the extent of their interaction with biological membranes. Ricin holotoxins containing the RTA mutants were, however, less cytotoxic to Vero cells than ricin containing wild-type RTA. At equivalent concentrations, a time course assay showed that holotoxin containing the mutant RTAs took longer to kill target cells than that containing wild-type recombinant RTA, suggesting that the mutant forms of RTA are less efficiently processed or translocated across an intracellular membrane than is wild-type RTA.
Protein Expression and Purification 11/1995; 6(5):665-70. · 1.59 Impact Factor
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ABSTRACT: Cytotoxic proteins that kill mammalian cells by catalytically inhibiting protein synthesis must enter the cytosol in order to reach their substrates. With the exception of diphtheria toxin, which enters the cytosol from acidified endosomes, the intracellular site of translocation of other toxins including ricin, Escherichia coli Shiga-like toxin-1, and Pseudomonas exotoxin A is likely to involve early compartments of the secretory pathway. We have used a molecular approach to identify the site and mechanism of toxin delivery to the cytosol by transiently expressing mutant GTPases that inhibit the assembly of biochemical complexes mediating anterograde and retrograde transport in the exocytic and endocytic pathways. The results provide evidence to suggest that receptors actively recycling between the endoplasmic reticulum and terminal Golgi compartments are essential for toxin translocation to the cytosol from the endoplasmic reticulum. The rapid kinetics of intoxication demonstrate a substantial level of bidirectional membrane flow and sorting through the early secretory pathway.
Journal of Biological Chemistry 09/1995; 270(34):20078-83. · 4.77 Impact Factor
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ABSTRACT: A series of mutations have been made in the carboxyl terminus of ricin A chain, centred on the hydrophobic region between amino acid residues Val245 and Val256. The mutant ricin A chains were expressed to a high level in an Escherichia coli system and the proteins purified to homogeneity. The enzymic activity of each of these A chain molecules was tested on rabbit reticulocyte ribosomes; in all cases, the activities were found to be comparable to wild-type recombinant ricin A chain. Following reassociation of these A chains to ricin B chain, Vero cells were challenged with these holotoxins and the cytotoxicities determined. Mutant ricin A chain with Ile247-->Ala was unable to reassociate and form holotoxin, indicating the importance of this residue in the interaction with ricin B chain. Mutant ricin A chain with Pro250-->Ala readily reassociated with ricin B chain, forming holotoxin with a 170-fold reduction in cytotoxicity to Vero cells. Other mutations in this region also produced A chain proteins which gave marked reductions in holotoxin cytotoxicity. We propose therefore that the C-terminal hydrophobic region of ricin A chain may be involved in membrane interactions prior to the translocation of this subunit into the cytosol, and that Pro250 plays a key role in one or both of these steps.
European Journal of Biochemistry 09/1995; 232(2):458-63. · 3.58 Impact Factor
