首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 31 毫秒
1.
Cytosolic components are required to produce the "primed donor" and to consume the "dilution-resistant" intermediates of the intercompartmental protein transport pathway as elucidated in a cell-free system (Balch, W. E., Glick, B. S., and Rothman, J. E. (1984) Cell 39, 525-536, and Wattenberg, B. W., Balch, W. E., and Rothman, J. E. (1986) J. Biol. Chem. 261, 2202-2207). Widely different levels of crude cytosol are required for each of these steps, suggesting that different cytosolic components might mediate each step. Here, we fractionate cytosol and demonstrate that there are multiple transport-active components. Furthermore, we report the development of stage-specific functional assays which reveal that a distinct soluble component is required in the consumption of the dilution-resistant intermediate. This component, of about 25 kilodaltons in its apparent native molecular mass, is derived from calf brain cytosol. While this component mediates the consumption of the dilution-resistant intermediate, it is inactive in the priming stage. This stage-specific component seems likely to be involved in the processing of transport vesicles after the attachment of those vesicles to the target membranes.  相似文献   

2.
Transport of a glycoprotein between compartments of the Golgi has been reconstituted in an in vitro system (Balch, W. E., Dunphy, W. G., Braell, W. A., and Rothman, J. E. (1984) Cell 39, 405-416). Cytosolic components and ATP are absolutely required for transport. Here, we have tested the acceptor activity of Golgi fractions and of cytosolic fractions prepared from a variety of organisms. All mammalian Golgi fractions can act as "acceptor" in the in vitro assay. Similarly, the cytosol fractions obtained from plants as well as animals and a lower eukaryote substitute for the homologous CHO cytosol normally used. Moreover, a cytosol subfraction prepared from wheat germ complements a different cytosolic fraction obtained from bovine brain. Apparently, the essential components involved in the post-translational protein transport are remarkably conserved between plants, animals, and lower eukaryotes.  相似文献   

3.
Semi-intact cells, a cell population in which the plasma membrane is perforated to expose intact intracellular organelles (Beckers, C. J. M., Keller, D. S., and Balch, W. E. (1987) Cell 50, 523-534), efficiently reconstitute vesicular trafficking of protein from the endoplasmic reticulum (ER) to the cis Golgi compartment. We now extend these studies to biochemically dissect transport of protein between the ER and the Golgi into a series of sequential intermediate steps involved in the budding and fusion of carrier vesicles. At least two broad categories of transport intermediates can be detected, those that involve early steps in transport and those involved in late, fusion-related events. Early transport steps require the transport of protein through a novel intermediate compartment in which protein accumulates at reduced temperature (15 degrees C). We demonstrate that both entry and exit from this 15 degrees C compartment can be successfully reconstituted in vitro. A late step in delivery of protein to the cis Golgi compartment requires Ca2+ (pCa7) and is coincident with a step which is sensitive to a peptide analog which blocks interaction between the Rab family of small GTP-binding proteins and a downstream effector protein(s) (Plutner, H., Schwaninger, R., Pind, S., and Balch, W. E. (1990) EMBO J. 9, 2375-2384). The combined results suggest that a single round of vesicular transport between the ER and the Golgi involves a rapid transit through N-ethylmaleimide-sensitive, guanosine 5'-(3-O-thio)triphosphate-sensitive, ATP- and cytosol-dependent step(s) involved in vesicle formation or transport to a novel intermediate compartment, followed by a regulated fusion event triggered in the presence of Ca2+ and functional components interacting with member(s) of the Rab gene family.  相似文献   

4.
Guanosine 5'-(3-O-thio)triphosphate (GTP gamma S), a non-hydrolyzable analogue of GTP, inhibits in vitro fusion among early endocytic vesicles in the presence of high concentrations of cytosol. In this report we show that fusion is remarkably stimulated by GTP gamma S under conditions where cytosolic components are the limiting factors for the process. The amount of cytosolic factors required for maximal fusion activity is several-fold decreased by the presence of GTP gamma S. Moreover, preincubation of vesicles in the presence of cytosol and GTP gamma S allows fusion to proceed even in the absence of cytosol. Our results indicate that a GTP-binding protein facilitates the binding of cytosolic factor(s) required for endosome fusion to the endosomal membrane and stabilizes a dilution-resistant intermediate of the fusion process.  相似文献   

5.
We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.  相似文献   

6.
Members of the Rab family of small molecular weight GTPases regulate the fusion of transport intermediates to target membranes along the biosynthetic and endocytic pathways. We recently demonstrated that Rab1 recruitment of the tethering factor p115 into a cis -SNARE complex programs coat protein II vesicles budding from the endoplasmic reticulum (donor compartment) for fusion with the Golgi apparatus (acceptor compartment) (Allan BB, Moyer BD, Balch WE. Science 2000; 289: 444–448). However, the molecular mechanism(s) of Rab regulation of Golgi acceptor compartment function in endoplasmic reticulum to Golgi transport are unknown. Here, we demonstrate that the cis -Golgi tethering protein GM130, complexed with GRASP65 and other proteins, forms a novel Rab1 effector complex that interacts with activated Rab1-GTP in a p115-independent manner and is required for coat protein II vesicle targeting/fusion with the cis -Golgi. We propose a 'homing hypothesis' in which the same Rab interacts with distinct tethering factors at donor and acceptor membranes to program heterotypic membrane fusion events between transport intermediates and their target compartments.  相似文献   

7.
The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide–sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5′-O-(3-thiotriphosphate) (GTPγS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPγS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPγS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.  相似文献   

8.
To understand intracellular trafficking modulations by live Salmonella, we investigated the characteristics of in vitro fusion between endosomes and phagosomes containing live (LSP) or dead Salmonella (DSP). We observed that fusion of both DSP and LSP were time, temperature and cytosol dependent. GTPgammaS and treatment of the phagosomes with Rab-GDI inhibited fusion, indicating involvement of Rab-GTPases. LSP were rich in rab5, alpha-SNAP, and NSF, while DSP mainly contained rab7. Fusion of endosomes with DSP was inhibited by ATP depletion, N-ethylmaleimide (NEM) treatment, and in NEM-sensitive factor (NSF)-depleted cytosol. In contrast, fusion of endosomes with LSP was not inhibited by ATP depletion or NEM treatment, and occurred in NSF-depleted cytosol. However, ATPgammaS inhibited both fusion events. Fusion of NEM-treated LSP with endosomes was abrogated in NSF- depleted cytosol and was restored by adding purified NSF, whereas no fusion occurred with NEM-treated DSP, indicating that NSF recruitment is dependent on continuous signals from live Salmonella. Binding of NSF with LSP required prior presence of rab5 on the phagosome. We have also shown that rab5 specifically binds with Sop E, a protein from Salmonella. Our results indicate that live Salmonella help binding of rab5 on the phagosomes, possibly activate the SNARE which leads to further recruitment of alpha-SNAP for subsequent binding with NSF to promote fusion of the LSP with early endosomes and inhibition of their transport to lysosomes.  相似文献   

9.
Tubular transport intermediates (TTIs) have been described as one class of transport carriers in endoplasmic reticulum (ER)-to-Golgi transport. In contrast to vesicle budding and fusion, little is known about the molecular regulation of TTI synthesis, transport and fusion with target membranes. Here we have used in vivo imaging of various kinds of GFP-tagged proteins to start to address these questions. We demonstrate that under steady-state conditions TTIs represent approximately 20% of all moving transport carriers. They increase in number and length when more transport cargo becomes available at the donor membrane, which we induced by either temperature-related transport blocks or increased expression of the respective GFP-tagged transport markers. The formation and motility of TTIs is strongly dependent on the presence of intact microtubules. Microinjection of GTPgammaS increases the frequency of TTI synthesis and the length of these carriers. When Rab proteins are removed from membranes by microinjection of recombinant Rab-GDI, the synthesis of TTIs is completely blocked. Microinjection of the cytoplasmic tails of the p23 and p24 membrane proteins also abolishes formation of p24-containing TTIs. Our data suggest that TTIs are ER-to-Golgi transport intermediates that form preferentially when transport-competent cargo exists in excess at the donor membrane. We propose a model where the interaction of the cytoplasmic tails of membrane proteins with microtubules are key determinants for TTI synthesis and may also serve as a so far unappreciated model for aspects of transport carrier formation.  相似文献   

10.
The assembly of many bacterial cell surface polysaccharides requires the transbilayer movement of polyisoprenoid-linked saccharide intermediates across the cytoplasmic membrane. It is generally believed that transverse diffusion of glycolipid intermediates is mediated by integral membrane proteins called translocases or "flippases." The bacterial genes proposed to encode these translocases have been collectively designated wzx genes. The wzxE gene of Escherichia coli K-12 has been implicated in the transbilayer movement of Fuc4NAc-ManNAcA-GlcNAc-P-P-undecaprenol (lipid III), the donor of the trisaccharide repeat unit in the biosynthesis of enterobacterial common antigen (ECA). Previous studies (Feldman, M. F., Marolda, C. L., Monteiro, M. A., Perry, M. B., Parodi, A. J., and Valvano, M. (1999) J. Biol. Chem. 274, 35129-35138) provided indirect evidence that the wzx(016) gene product of E. coli K-12 encoded a translocase capable of mediating the transbilayer movement of N-acetylglucosaminylpyrophosphorylundecaprenol (GlcNAc-P-P-Und), an early intermediate in the synthesis of ECA and many lipopolysaccharide O antigens. Therefore, genetic and biochemical studies were conducted to determine if the putative Wzx(O16) translocase was capable of mediating the transport of N-acetylglucosaminylpyrophosphorylnerol (GlcNAc-P-P-Ner), a water-soluble analogue of GlcNAc-P-P-Und. [(3)H]GlcNAc-P-P-Ner was transported into sealed, everted cytoplasmic membrane vesicles of E. coli K-12 as well as a deletion mutant lacking both the wzx(016) and wzxC genes. In contrast, [(3)H]GlcNAc-P-P-Ner was not transported into membrane vesicles prepared from a wzxE-null mutant, and metabolic radiolabeling experiments revealed the accumulation of lipid III in this mutant. The WzxE transport system exhibited substrate specificity by recognizing both a pyrophosphoryl-linked saccharide and an unsaturated alpha-isoprene unit in the carrier lipid. These results support the conclusion that the wzxE gene encodes a membrane protein involved in the transbilayer movement of lipid III in E. coli.  相似文献   

11.
The lysosomotropic amine primaquine has previously been shown to inhibit both secretory and recycling processes of cells in culture. We have used a cell-free assay that reconstitutes glycoprotein transport through the Golgi apparatus to investigate the mechanism of action of primaquine. In this assay, primaquine inhibits protein transport at a half-maximal concentration of 50 microM, similar to the concentration previously reported to disrupt protein secretion in cultured cells. Kinetic analysis of primaquine inhibition indicates that its point of action is at an early step in the vesicular transport mechanism. Primaquine does not inhibit the fusion of vesicles already attached to their target membranes. Primaquine irreversibly inactivates the membranes that form transport vesicles (donor), but not the membranes that are the destination of those vesicles (acceptor). Morphological data indicate that primaquine inhibits the budding of vesicles from the donor membranes. Once formed, the vesicles are refractile to primaquine action, and their attachment to and fusion with acceptor membranes proceeds unimpeded. In addition to illuminating the mechanism of action of primaquine, this study suggests that the selective action of this agent will make it a useful tool in the study of the formation of transport vesicles.  相似文献   

12.
Soya N  Fang Y  Palcic MM  Klassen JS 《Glycobiology》2011,21(5):547-552
The enzymatic mechanism by which retaining glycosyltransferases (GTs) transfer monosaccharides with net retention of the anomeric configuration has, so far, resisted elucidation. Here, direct detection of covalent glycosyl-enzyme intermediates for mutants of two model retaining GTs, the human blood group synthesizing α-(1 → 3)-N-acetylgalactosaminyltransferase (GTA) and α-(1 → 3)-galactosyltransferase (GTB) mutants, by mass spectrometry (MS) is reported. Incubation of mutants of GTA or GTB, in which the putative catalytic nucleophile Glu(303) was replaced with Cys (i.e. GTA(E303C) and GTB(E303C)), with their respective donor substrate results in a covalent intermediate. Tandem MS analysis using collision-induced dissociation confirmed Cys(303) as the site of glycosylation. Exposure of the glycosyl-enzyme intermediates to a disaccharide acceptor results in the formation of the corresponding enzymatic trisaccharide products. These findings suggest that the GTA(E303C) and GTB(E303C) mutants may operate by a double-displacement mechanism.  相似文献   

13.
Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex. To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate. A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes. Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate. Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.  相似文献   

14.
Transport of the vesicular stomatitis virus (VSV)-encoded glycoprotein (G protein) between successive compartments of the Golgi in a cell-free system is measured by the coupled incorporation of N-[3H]acetylglucosamine (GlcNAc). This glycosylation occurs when G protein is transported from a "donor" compartment in Golgi membranes that lack GlcNAc transferase I (from VSV-infected CHO clone 15B cells) to the next "acceptor" compartment in a Golgi population from wild-type CHO cells (containing the GlcNAc transferase but not G protein). Here we present a detailed characterization of the conditions required to achieve transport in vitro. We find that donor and acceptor activities differ markedly in certain of their properties. The donor activity is inhibited by N-ethylmaleimide but the acceptor activity is resistant. Donor activity is unstable in the absence of ATP or the cytosol fraction; acceptor activity is much more stable. This asymmetry may reflect the vectorial nature of the underlying biochemistry of protein transport. Both donor and acceptor are trypsin-sensitive, implying a need for cytoplasmically oriented membrane proteins. Transport occurs only in a restricted range of close to physiological conditions. ATP is absolutely required, although as little as 1 microM is sufficient. Transport is inhibited by ATP-gamma-sulfate and vanadate, suggesting that ATP hydrolysis is needed. By contrast, ionophores that dissipate membrane potentials and proton gradients do not inhibit transport. Monensin was also without effect in the cell-free system.  相似文献   

15.
The mechanisms of vesicle budding and fusion   总被引:45,自引:0,他引:45  
Bonifacino JS  Glick BS 《Cell》2004,116(2):153-166
Genetic and biochemical analyses of the secretory pathway have produced a detailed picture of the molecular mechanisms involved in selective cargo transport between organelles. This transport occurs by means of vesicular intermediates that bud from a donor compartment and fuse with an acceptor compartment. Vesicle budding and cargo selection are mediated by protein coats, while vesicle targeting and fusion depend on a machinery that includes the SNARE proteins. Precise regulation of these two aspects of vesicular transport ensures efficient cargo transfer while preserving organelle identity.  相似文献   

16.
17.
Recently, it has become clear that the influenza virus fusion protein, hemagglutinin (HA), produces membrane destabilization and fusion by a multistep process, which involves the aggregation of the HAs to form a fusion site. While the details of this process are under debate, it is important to recognize that proposing any sequence of "microscopic" fusion intermediates encumbers general "macroscopic" kinetic consequences, i.e., with respect to membrane mixing rates. Using a kinetic scheme which incorporates the essential elements of several recently proposed models, some of these measurable properties have been elucidated. First, a rigorous mathematical relationship between fusion intermediates and the fusion event itself is defined. Second, it is shown that what is measured as the macroscopic "fusion rate constant" is a simple function of all of the rate constants governing the transitions between intermediates, whether or not one of the microscopic steps is rate limiting. Third, while this kinetic scheme predicts a delay (or lag) time for fusion, as has been observed, it will be very difficult to extract reliable microscopic information from these data. Furthermore, it is predicted that the delay time can depend upon HA surface density even when the HA aggregation step is very rapid compared with fusion, i.e., the delay time need not be due to HA aggregation. Fourth, the inactivation process observed for influenza virions at low pH can be described within this kinetic scheme simply, yet rigorously, via the loss of the fusion intermediates. Fifth, predicted Arrhenius plots of fusion rates can be linear for this multistep scheme, even though there is no single rate-determining step and even when a branched step is introduced, i.e., where one pathway predominates at low temperature and the other pathway predominates at high temperature. Furthermore, the apparent activation energies obtained from these plots bear little or no quantitative resemblance to the microscopic activation energies used to simulate the data. Overall, these results clearly show that the intermediates of protein mediated fusion can be studied only by using assays sensitive to the formation of each proposed intermediate.  相似文献   

18.
The Semliki Forest virus (SFV) glycoprotein precursor p62 is processed to the E2 and E3 during the transport from the trans-Golgi network (TGN) to the cell surface. We have studied the regulation of the membrane fusion machinery (Rab/N-ethylmaleimide (NEM)-sensitive fusion protein (NSF)/soluble NSF attachment protein (SNAP)-SNAP receptor) in this processing. Activation of the disassembly of this complex with recombinant NSF stimulated the cleavage of p62 in permeabilized cells. Inactivation of NSF with a mutant alpha-SNAP(L294A) or NEM treatment inhibited processing of p62. Rab GDP dissociation inhibitor inhibited the cleavage. Inactivation of NSF blocks the transport of SFV glycoproteins and vesicular stomatitis virus G-glycoprotein from the TGN membranes to the cell surface. The results support the conclusion that inhibition of membrane fusion arrests p62 in the TGN and prevents its processing by furin.  相似文献   

19.
Crude extracts of Xenopus eggs are capable of nuclear assembly around chromatin templates or even around protein-free, naked DNA templates. Here the requirements for nuclear assembly around a naked DNA template were investigated. Extracts were separated by ultracentrifugation into cytosol, membrane, and gelatinous pellet fractions. It was found that, in addition to the cytosolic and membrane fractions, a component of the gelatinous pellet fraction was required for the assembly of functional nuclei around a naked DNA template. In the absence of this component, membrane-bound but functionally inert spheres of lambda DNA were formed. Purification of the active pellet factor unexpectedly demonstrated the component to be glycogen. The assembly of functionally active nuclei, as assayed by DNA replication and nuclear transport, required that glycogen be pre-incubated with the lambda DNA and cytosol during the period of chromatin and higher order intermediate formation, before the addition of membranes. Hydrolysis of glycogen with alpha- amylase in the extract blocked nuclear formation. Upon analysis, chromatin formed in the presence of cytosol and glycogen alone appeared highly condensed, reminiscent of the nuclear assembly intermediate described by Newport in crude extracts (Newport, J. 1987. Cell. 48:205- 217). In contrast, chromatin formed from phage lambda DNA in cytosol lacking glycogen formed "fluffy chromatin-like" structures. Using sucrose gradient centrifugation, the highly condensed intermediates formed in the presence of glycogen could be isolated and were now able to serve as nuclear assembly templates in extracts lacking glycogen, arguing that the requirement for glycogen is temporally restricted to the time of intermediate formation and function. Glycogen does not act simply by inducing condensation of the chromatin, since similarly isolated mitotically condensed chromatin intermediates do not form functional nuclei. However, both mitotic and fluffy interphase chromatin intermediates formed in the absence of glycogen can be rescued to form functional nuclei when added to a second extract which contains glycogen. This study presents a novel role for a carbohydrate in nuclear assembly, a role which involves the formation of a particular chromatin intermediate. Potential models for the role of glycogen are discussed.  相似文献   

20.
During ubiquitin conjugation, the thioester bond that links "donor" ubiquitin to ubiquitin-conjugating enzyme (E2) undergoes nucleophilic attack by the ?-amino group of an acceptor lysine, resulting in formation of an isopeptide bond. Models of ubiquitination have envisioned the donor ubiquitin to be a passive participant in this process. However, we show here that the I44A mutation in ubiquitin profoundly inhibits its ability to serve as a donor for ubiquitin chain initiation or elongation, but can be rescued by computationally predicted compensatory mutations in the E2 Cdc34. The donor defect of ubiquitin-I44A can be partially suppressed either by using a low pKa amine (hydroxylamine) as the acceptor or by performing reactions at higher pH, suggesting that the discharge defect arises in part due to inefficient deprotonation of the acceptor lysine. We propose that interaction between Cdc34 and the donor ubiquitin organizes the active site to promote efficient ubiquitination of substrate.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号