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Membranes and sorting
475
Membranes and sorting Editorial overview Scott D Emr* and Vivek Malhotrat Addresses *Division of Cellular and Molecular Medicine, and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 92093-0668, USA iDepartment of Biology, University of California at San Diego, La Jolla, CA 92093-0347, USA Current Opinion in Cell Biology 1997, 9:475-476 http:Nbiomednet.comlelecreflOQ55067400900475 0 Current Biology Ltd ISSN 0955-0674 Abbreviations AP adaptor protein NSF N-ethylmaleimide-sensitive factor SNARE soluble NSF attachment protein receptor
Cargo loading and vesicle-mediated
transport
Significant progress has been made during the past few years in the identification of the molecular machinery that directs vesicle-mediated transport from one station to another in the secretory or endocytic systems. This progress has been largely driven by the fact that biochemical, genetic and molecular approaches used to study divergent cell types (e.g. yeast and mammalian neurons) have converged on a common set of transport components. It seems that, with the discovery of each new component, we are only too willing to propose new models and paradigms to accommodate these novel factors. Thus, our enthusiasm is now being tempered by the realization that our early hypotheses do not explain many of the most recent observations, resulting in some healthy controversies and revisions to our favorite models. The transport process in principle ‘simply’ requires the packaging of cargo into a vesicle carrier and the transport, docking and fusion of the vesicle intermediate with the appropriate target membrane. Unexpectedly, this set of biochemical reactions depends on a considerable array of proteins, lipids and enzyme complexes (coats, SNARES [soluble NSF attachment protein receptors], GTPases, ATPases, kinases, phosphoinositides, etc.), some of which function as structural components while others catalyze the assembly/disassembly of reaction intermediates or regulate spatial and temporal aspects of the process. In this section of Curyerjt Opitriotr itr Cell Biology, we have assembled a number of significant reviews on major topics pertaining to vesicle formation, docking and fusion. Instead of providing a general overview of what is discussed in these reviews, we provide a summary of some of the recent advances and controversies in the field.
Cargo packaging
Bonifacino and Riezman (pp - _ 488-495) deal with this area of research. During many stages of intercompartmental transport, membrane and lumenal proteins are sorted into budding vesicles. It is reasonable to predict that, in the case of integral membrane cargo proteins, there is a direct interaction with the vesicle coat components. This is well known for plasma membrane specific receptors, which can interact through their tyrosine-based motifs with the cytoplasmic components of the coats (adaptor proteins AP-1 and AP-2). Similarly, integral membrane proteins in intracellular compartments interact via their tails through tyrosine-, diphenylalanine-, dileucine- and dilysine-based motifs with components of the clathrin and non-clathrin (COP1 and COPII) coats. But the problem arises when one considers the packaging of soluble cargo into the budding vesicles. On the basis of analogy with the mannose 6-phosphate receptor, it is reasonable to assume that the soluble cargo interacts with an integral membrane protein, which in turn carries a specific motif for binding the cytoplasmically oriented coat components (see the review by Traub and Kornfeld, pp 527-533). It would be difficult to predict that each soluble cargo molecule has a specific membrane receptor and the more likely scenario is that the proposed membrane receptors will recognize a motif or modification that is common to soluble cargo targeted to a common destination. Possible membrane receptors that have attracted attention in recent years are the members of the Emp24 family of membrane proteins and the lectin-like proteins, for example ERGIC53. Deletion of individual members of the Emp24 family has not yet uncovered a dramatic defect in cargo transport. The question of soluble cargo selection into the budding vesicle therefore remains a challenge.
Coat recruitment
and vesicle fission
This brings us to the next question: what regulates or triggers vesicle formation from a given compartment? Is the accumulation of the cargo a signal for the membranes to activate the process that ultimately generates a vesicle? And what role do membrane lipids and cytoplasmic components, such as coats and small GTPases like ARF (ADP-ribosylation factor) and Rab proteins, play in the process of vesicle formation! Are the coats involved in molding the membranes into a bud or do they serve an alternative function of merely selecting the cargo for export? The reviews by Roth and Sternweis (pp 519-526) and by Harder and Simons (pp 534-542) address these questions and provide insights into the roles that coats, protein-lipid interactions and lipid-lipid interactions play in vesicle formation and cargo loading.
and vesicle coats
The reviews by Kuehn and Schekman (pp 477483), Cosson and Letourneur (pp 484487) and Kirchhausen,
Another major point regarding be addressed is the mechanism
the coats that needs to by which these compo-
476
Membranes and sorting
nents are recruited to distinct intracellular membranes. Transmembrane proteins containing tyrosine-based sorting motifs are found in the Golgi, endosomes and the plasma membrane, yet the components with which they interact appear to be different at each membrane. For example, the tyrosine-based motifs present in the cytoplasmic tails of proteins in the Golgi membrane interact with the cytoplasmic adaptor protein AP-1 while the same tyrosine motif in a plasma membrane protein interacts with AP-2. How can the same sorting motif bind to distinct transport components? Selective docking sites must be present on each membrane to recruit the appropriate cytoplasmic transport factors. Because integral membrane proteins that ultimately reside in different intracellular compartments all have their origins in the endoplasmic reticulum, certain sorting signals presumably remain silent until the proteins encounter the appropriate coat components that bind and sort these proteins to their correct site of function. The identification of the mechanism responsible for specific recruitment of cytoplasmic coat components to a given target membrane is, therefore, crucial to our understanding of the sorting and stage-specific packaging of the cargo. It still remains to be determined what causes the final fission of the bud to generate a vesicle. In the case of clathrin-coated endocytic vesicles, the protein dynamin appears to facilitate the final events in vesicle budding. However, dynamin alone is not likely to be sufficient for this process and it is also noteworthy that dynamin-like proteins have not been found to be associated with COPI- or COPII-coated vesicles.
Vesicle docking and membrane fusion Once the cargo-containing vesicles have pinched off from the donor organelle membrane, they must be targeted to and fuse with the correct destination membrane. Members of the SNARE, ,Rab and Secl families of proteins appear to direct and regulate these vesicle docking and fusion reactions (see reviews by Novick and Zerial, pp 496-504, and Hay and Scheller, pp 505-512). However, recent work questions the role of SNARE proteins (vesicle [VI-SNARES and target membrane [t]-SNARES) as well as of Rab proteins in determining the inherent specificity of vesicle-docking reactions. The precise role of the Secl proteins also remains elusive. Other, as yet uncharaccerized, factors may ultimately be required to ensure that each transport vesicle docks and fuses with a single destination compartment. The reviews by Hay and Scheller (pp 505-512) and Goda and Siidhof (pp 513-518) provide more recent insights into the potential roles of these components. Many questions still remain unanswered regarding vesicle docking/fusion. Because V-SNARES and other membrane proteins must be recycled back to the donor compartment, what distinguishes the forward vesicles from the recycling vesicles that carry the same proteins but are directed towards a different target compartment? One other major concern that needs to be resolved at this stage is the
distinction between components involved in docking versus fusion events. It is also important to acknowledge that the regulation of the components of the so-called ‘SNARE complex’ will be different depending on the source of the fusing membrane partners. For example, on the basis of the intra-Golgi transport assay, it has been widely acknowledged that NSF acts at a late stage in the fusion process, probably after docking, and that ATP hydrolysis by NSF leads to changes in the docked membranes that subsequently lead to fusion. More recent studies, however, provide new roles for NSF. Based on work in endocrine and neuronal cells, it is known that ATP is consumed in the priming reaction prior to calcium-triggered membrane fusion, indicating that NSF-based ATP hydrolysis is necessary prior to the fusion events. In addition, in vitro assays reconstituting fusion between isolated yeast vacuoles indicate that NSF is necessary but that its function is carried out even before docking of the fusing partners. Of course, if the sole function of NSF is to disassemble the paired SNARES, then it makes sense that, in the case of homotypic fusion, the SNARES can be separated prior to docking, thus fulfilling the requirement for NSF However, in the case of heterotypic fusion, the SNARES pair only after docking and hence the requirement of NSF for this late step.
Perspectives In the face of recent progress, numerous additional challenging questions still remain to be addressed. We have not, for example, touched on post-docking events. Recent studies have revealed the identity of new proteins (e.g. the exocyst fusion complex) that are required for a late stage of secretory vesicle docking/fusion with the plasma membrane in yeast and human cells. What role do these proteins play? Are they involved in preparing the acceptor membrane for the receipt of incoming vesicles, are they involved in priming of the t-SNARES, or do they play a role in post-SNARE fusion events? This brings us to the lack of an absolute requirement for the SNARES or Rab proteins in at least some transport steps. Recent experiments indicate that the function of individual SNARES or Rab proteins can be bypassed, suggesting that these functions may not be absolutely required for vesicle-mediated transport (see Novick and Zerial, pp 496-504). If not SNARES and Rab proteins, then which proteins are essential? What regulates the number of vesicles that bud from a given compartment, and how is the coupling between anterograde versus retrograde transport between pairs of compartments regulated? Answers to these questions may provide insights into the more global issues of maintenance of the compartmental boundaries between secretory and endocytic organelles amidst the enormous flux through them. Undoubtedly, the list of questions will increase as we learn more about the complexity of the membrane-trafficking process and its relationship with the overall organization of the secretory and endocytic systems.
Membranes and sorting Editorial overview Scott D Emr* and Vivek Malhotrat Addresses *Division of Cellular and Molecular Medicine, and Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, CA 92093-0668, USA iDepartment of Biology, University of California at San Diego, La Jolla, CA 92093-0347, USA Current Opinion in Cell Biology 1997, 9:475-476 http:Nbiomednet.comlelecreflOQ55067400900475 0 Current Biology Ltd ISSN 0955-0674 Abbreviations AP adaptor protein NSF N-ethylmaleimide-sensitive factor SNARE soluble NSF attachment protein receptor
Cargo loading and vesicle-mediated
transport
Significant progress has been made during the past few years in the identification of the molecular machinery that directs vesicle-mediated transport from one station to another in the secretory or endocytic systems. This progress has been largely driven by the fact that biochemical, genetic and molecular approaches used to study divergent cell types (e.g. yeast and mammalian neurons) have converged on a common set of transport components. It seems that, with the discovery of each new component, we are only too willing to propose new models and paradigms to accommodate these novel factors. Thus, our enthusiasm is now being tempered by the realization that our early hypotheses do not explain many of the most recent observations, resulting in some healthy controversies and revisions to our favorite models. The transport process in principle ‘simply’ requires the packaging of cargo into a vesicle carrier and the transport, docking and fusion of the vesicle intermediate with the appropriate target membrane. Unexpectedly, this set of biochemical reactions depends on a considerable array of proteins, lipids and enzyme complexes (coats, SNARES [soluble NSF attachment protein receptors], GTPases, ATPases, kinases, phosphoinositides, etc.), some of which function as structural components while others catalyze the assembly/disassembly of reaction intermediates or regulate spatial and temporal aspects of the process. In this section of Curyerjt Opitriotr itr Cell Biology, we have assembled a number of significant reviews on major topics pertaining to vesicle formation, docking and fusion. Instead of providing a general overview of what is discussed in these reviews, we provide a summary of some of the recent advances and controversies in the field.
Cargo packaging
Bonifacino and Riezman (pp - _ 488-495) deal with this area of research. During many stages of intercompartmental transport, membrane and lumenal proteins are sorted into budding vesicles. It is reasonable to predict that, in the case of integral membrane cargo proteins, there is a direct interaction with the vesicle coat components. This is well known for plasma membrane specific receptors, which can interact through their tyrosine-based motifs with the cytoplasmic components of the coats (adaptor proteins AP-1 and AP-2). Similarly, integral membrane proteins in intracellular compartments interact via their tails through tyrosine-, diphenylalanine-, dileucine- and dilysine-based motifs with components of the clathrin and non-clathrin (COP1 and COPII) coats. But the problem arises when one considers the packaging of soluble cargo into the budding vesicles. On the basis of analogy with the mannose 6-phosphate receptor, it is reasonable to assume that the soluble cargo interacts with an integral membrane protein, which in turn carries a specific motif for binding the cytoplasmically oriented coat components (see the review by Traub and Kornfeld, pp 527-533). It would be difficult to predict that each soluble cargo molecule has a specific membrane receptor and the more likely scenario is that the proposed membrane receptors will recognize a motif or modification that is common to soluble cargo targeted to a common destination. Possible membrane receptors that have attracted attention in recent years are the members of the Emp24 family of membrane proteins and the lectin-like proteins, for example ERGIC53. Deletion of individual members of the Emp24 family has not yet uncovered a dramatic defect in cargo transport. The question of soluble cargo selection into the budding vesicle therefore remains a challenge.
Coat recruitment
and vesicle fission
This brings us to the next question: what regulates or triggers vesicle formation from a given compartment? Is the accumulation of the cargo a signal for the membranes to activate the process that ultimately generates a vesicle? And what role do membrane lipids and cytoplasmic components, such as coats and small GTPases like ARF (ADP-ribosylation factor) and Rab proteins, play in the process of vesicle formation! Are the coats involved in molding the membranes into a bud or do they serve an alternative function of merely selecting the cargo for export? The reviews by Roth and Sternweis (pp 519-526) and by Harder and Simons (pp 534-542) address these questions and provide insights into the roles that coats, protein-lipid interactions and lipid-lipid interactions play in vesicle formation and cargo loading.
and vesicle coats
The reviews by Kuehn and Schekman (pp 477483), Cosson and Letourneur (pp 484487) and Kirchhausen,
Another major point regarding be addressed is the mechanism
the coats that needs to by which these compo-
476
Membranes and sorting
nents are recruited to distinct intracellular membranes. Transmembrane proteins containing tyrosine-based sorting motifs are found in the Golgi, endosomes and the plasma membrane, yet the components with which they interact appear to be different at each membrane. For example, the tyrosine-based motifs present in the cytoplasmic tails of proteins in the Golgi membrane interact with the cytoplasmic adaptor protein AP-1 while the same tyrosine motif in a plasma membrane protein interacts with AP-2. How can the same sorting motif bind to distinct transport components? Selective docking sites must be present on each membrane to recruit the appropriate cytoplasmic transport factors. Because integral membrane proteins that ultimately reside in different intracellular compartments all have their origins in the endoplasmic reticulum, certain sorting signals presumably remain silent until the proteins encounter the appropriate coat components that bind and sort these proteins to their correct site of function. The identification of the mechanism responsible for specific recruitment of cytoplasmic coat components to a given target membrane is, therefore, crucial to our understanding of the sorting and stage-specific packaging of the cargo. It still remains to be determined what causes the final fission of the bud to generate a vesicle. In the case of clathrin-coated endocytic vesicles, the protein dynamin appears to facilitate the final events in vesicle budding. However, dynamin alone is not likely to be sufficient for this process and it is also noteworthy that dynamin-like proteins have not been found to be associated with COPI- or COPII-coated vesicles.
Vesicle docking and membrane fusion Once the cargo-containing vesicles have pinched off from the donor organelle membrane, they must be targeted to and fuse with the correct destination membrane. Members of the SNARE, ,Rab and Secl families of proteins appear to direct and regulate these vesicle docking and fusion reactions (see reviews by Novick and Zerial, pp 496-504, and Hay and Scheller, pp 505-512). However, recent work questions the role of SNARE proteins (vesicle [VI-SNARES and target membrane [t]-SNARES) as well as of Rab proteins in determining the inherent specificity of vesicle-docking reactions. The precise role of the Secl proteins also remains elusive. Other, as yet uncharaccerized, factors may ultimately be required to ensure that each transport vesicle docks and fuses with a single destination compartment. The reviews by Hay and Scheller (pp 505-512) and Goda and Siidhof (pp 513-518) provide more recent insights into the potential roles of these components. Many questions still remain unanswered regarding vesicle docking/fusion. Because V-SNARES and other membrane proteins must be recycled back to the donor compartment, what distinguishes the forward vesicles from the recycling vesicles that carry the same proteins but are directed towards a different target compartment? One other major concern that needs to be resolved at this stage is the
distinction between components involved in docking versus fusion events. It is also important to acknowledge that the regulation of the components of the so-called ‘SNARE complex’ will be different depending on the source of the fusing membrane partners. For example, on the basis of the intra-Golgi transport assay, it has been widely acknowledged that NSF acts at a late stage in the fusion process, probably after docking, and that ATP hydrolysis by NSF leads to changes in the docked membranes that subsequently lead to fusion. More recent studies, however, provide new roles for NSF. Based on work in endocrine and neuronal cells, it is known that ATP is consumed in the priming reaction prior to calcium-triggered membrane fusion, indicating that NSF-based ATP hydrolysis is necessary prior to the fusion events. In addition, in vitro assays reconstituting fusion between isolated yeast vacuoles indicate that NSF is necessary but that its function is carried out even before docking of the fusing partners. Of course, if the sole function of NSF is to disassemble the paired SNARES, then it makes sense that, in the case of homotypic fusion, the SNARES can be separated prior to docking, thus fulfilling the requirement for NSF However, in the case of heterotypic fusion, the SNARES pair only after docking and hence the requirement of NSF for this late step.
Perspectives In the face of recent progress, numerous additional challenging questions still remain to be addressed. We have not, for example, touched on post-docking events. Recent studies have revealed the identity of new proteins (e.g. the exocyst fusion complex) that are required for a late stage of secretory vesicle docking/fusion with the plasma membrane in yeast and human cells. What role do these proteins play? Are they involved in preparing the acceptor membrane for the receipt of incoming vesicles, are they involved in priming of the t-SNARES, or do they play a role in post-SNARE fusion events? This brings us to the lack of an absolute requirement for the SNARES or Rab proteins in at least some transport steps. Recent experiments indicate that the function of individual SNARES or Rab proteins can be bypassed, suggesting that these functions may not be absolutely required for vesicle-mediated transport (see Novick and Zerial, pp 496-504). If not SNARES and Rab proteins, then which proteins are essential? What regulates the number of vesicles that bud from a given compartment, and how is the coupling between anterograde versus retrograde transport between pairs of compartments regulated? Answers to these questions may provide insights into the more global issues of maintenance of the compartmental boundaries between secretory and endocytic organelles amidst the enormous flux through them. Undoubtedly, the list of questions will increase as we learn more about the complexity of the membrane-trafficking process and its relationship with the overall organization of the secretory and endocytic systems.