- Email: [email protected]
Membranes and sorting
Membranes and sorting Editorial overview Jean Cruenberg and Thomas E Kreis University
Current
of Geneva,
Opinion
Geneva,
in Cell Biology
As the various events during the journey of a membrane or secretory protein from its site of synthesis, the endoplasmic reticulum (ER), to its final destination (e.g. the plasma membrane), continue to be unraveled, and ‘Membranes and sorting’ issues of Current Opinion in Cell Biology pass by, surprising findings are reported at regular intervals. Two of the most recent highlights of the past year’s discoveries indicate an additional unexpected function for the COPI coat proteins in retrieval of ER membrane proteins and the characterization of COPII, a third class of a transport vesicle associated coat essential for membrane traflic from the ER to the Golgi complex in yeast. The prospect that a family of coat proteins, related in structure and function, regulates all the different steps of intracellular membrane traffic appears to be becoming a reality The basis of this rapid pace of progress in understanding membrane biogenesis and cellular organization is founded on the combination of cellular genetics, molecular cell biology, biochemical reconstitution of defined processes in cell-free systems, and manipulation of living cells. We believe that a flavor of the power of this multidisciplinary approach is illustrated by the contributions in this issue on ‘Membranes and sorting’ by prominent scholars in the field. Helenius and his colleagues are deciphering the fine mechanisms of folding and maturation of membrane and secretory proteins (Hammond and Helenius, pp 523-529). An elaborate system of factors (including calnexin, calreticulin, ERGIC53/p58, BiP and other ER chaperones) is being unraveled, which ensures that only the correctly modified and properly folded proteins progress along the assembly line between the ER and Golgi complex and finally leave the early compartment(s) of biosynthetic membrane transport. Several of these factors are lectins that can discriminate between the different states of carbohydrate trimming on the transported proteins. In fact, a model is proposed in which these factors responsible for quality control build up an extended network or retention matrix that regulates exit from this first station of synthesis and transport to the next compartment.
Switzerland
1995,
7:519-522
ER proteins that have left their site of original function, for example because they are chaperoning newly synthesized protein to the next station in the biosynthetic pathway or because they have been missorted or escaped an ER retention mechanism, must be retrieved to ensure maximal efficiency of the system. Signals and some of the mechanisms that lead to retention or retrieval of proteins in the ER have been identified and characterized in detail (Pelham, pp 530-535). A recent exciting observation, also discussed in this article, implicates COPI, a coat associated with transport vesicles between the intermediate compartment and the Golgi complex, with retrieval of ER proteins containing the di-lysine ER-retention motif (KKXX in the single-letter code for amino acids; X is any amino acid). Biochemical evidence for direct interaction of polypeptides containing KKXX at their carboxyl terminus with COP1 has been obtained, and a genetical approach in yeast lead to the molecular characterization of U-COP (Retlp). The boundaries between the well defined rough ER and the cis-cisternae of the (medial) Golgi stack are so far only poorly defined. A compartment located between the ER and the Golgi complex has been proposed to exist and has been given many names (we use intermediate compartment in this overview). In addition, a network of tubular-cisternal structures, the cis-Golgi network, appears to be the port of entry for material destined to reach and transit through the cisternae of the Golgi complex. So far, the identity of both the intermediate compartment and the &Golgi network remains obscure, as no distinct membrane-bounded compartment has not been clearly defined between the rough ER and the stacked Golgi cisternae, presumably because of the highly dynamic nature of the membranes in this zone. In fact, continuity of ER membranes with the &most cisterna of the Golgi stack has recently been proposed by Gareth Grifiths and his colleagues [l]. It is essential to know the precise number and arrangement of the membrane-bounded compartments in the early biosynthetic membrane pathway, as these
Abbreviations ER-endoplasmic
reticulum; SNARE-SNAP
receptor; t-SNARE-target
SNARE; v-SNARE-vesicle
0 Current Biology Ltd ISSN 0955-0674
SNARE.
519
520
Membranes and sorting
parameters define the minimal number of vesicular steps required for transfer of cargo. So far, two classes of vesicle-associated coats implicated in membrane traffic between the ER and the Golgi complex have been characterized: COPI and, more recently, COP11 (Salama and Schekman, pp 536-543). The precise functions and steps of action of these two coat proteins are still enigmatic and thus different possible models are discussed. While COP11 appears to be involved exclusively in anterograde transport from the ER, the role of COPI may be more complex. Given the interaction of COP1 with some of the membrane proteins of the quality control machinery (i.e. proteins with KKXX ER-retention/retrieval signals), COP1 may be associated with shuttling of tranport intermediates or recycling the factors essential for assembly of membrane proteins until they are ready to reach the stacked cisternae of the Golgi complex. Transport through the cisternae of the medial Golgi has been studied and discussed in great detail [2]. Although several aspects in the current model describing membrane traffic through that organelle still need further clarification (for example, see [3,4]), reconstitution of this transport in vitro led to the discovery and molecular characterization of several ubiquitous key factors regulating membrane traffic. At the exit site of the Golgi complex, the trans-Golgi network, proteins are sorted into different vesicular carriers and delivered to their final destination. The example described in this issue addresses protein transport from the late Golgi, presumably the truns-Golgi network, to the yeast vacuole (Horazdovsky, DeWald and Emr, pp 544-551). Protein trafficking to the vacuole depends on a set of proteins whose homologues are also known to be implicated in other steps of vesicular transport, in yeast or mammalian cells. These include a small GTP-binding protein homologous to Rab5 (Vps2lp/Ypt5lp), a dynamin-like GTPase (Vpslp), and Seclp homologues (vps45p and Vps33p). Of particular interest is their finding that transport to the vacuole also depends on mositol 3-kinase (Vps34p). Membrane a phosphatid y 1’ association and activation of Vps34p appears to be mediated by a serine/threonine protein kinase, Vpsl5p. Several mechanisms by which vesicular traffic may be regulated via phosphoinositides are discussed. In their review, Lamaze and Schmid (pp 573-580) discuss the different routes of entry into animal cells. Receptor-mediated endocytosis via clathrin-coated pits and vesicles remains the best characterized endocytic pathway, and is probably the major route of plasma membrane protein and lipid turnover in most cell types. In vitro studies by Schmid and colleagues, together with the morphological studies of de Camilli and his group, now suggest that polymerization of the GTPase dynamin at the neck of an invaginated pit provides the molecular device which constricts the neck until it is severed from the membrane. Interestingly, uptake of solutes resumes after inhibition of the clathrin pathway
in a dynamin temperature-sensitive mutant, suggesting that an alternate pathway can compensate for the loss of the clathrin pathway. These observations agree with earlier observations indicating that an additional endocytic pathway leading to endosomes must exist. A third pathway, mediated by caveolae, has also been postulated. Studies carried out by Parton et al. [5] now unambiguously show that caveolae can detach from the cell surface, and form intracellular vesicles, albeit at a very low rate when compared to clathrin-coated vesicles. The intracellular destination of these vesicles, however, is still being debated. The cover of this issue illustrates the presence of a ‘bunch’ of these vesicles at the plasma membrane, emphasizing the intriguing complexity of the organization of caveolae in some cell types. The functional analogy between endocytosis in yeast and mammalian cells remains an important question. In yeast cells, studies from the groups of Riezman and Botstein indicate that endocytosis may be actin-driven, in contrast to clathrin-mediated internalization in mammalian cells. Homologues of molecules implicated in clathrin-mediated endocytosis in mammalian cells appear, in fact, to be necessary for vacuolar protein sorting in yeast (Rab5 and dynamin; see below and Horazdovsky, DeWald and Emr). Thus, Golgi-to-endosome transport in yeast has mechanistic similarities to clathrin-mediated internalization in mammalian cells. Gruenberg and Maxfield (pp 552-563) discuss membrane transport in the endocytic pathway. A comparison between in vivo and in vitro observations indicates that both early and late endosomes are highly dynamic organelles, connected by transport intermediates. These intermediates probably correspond to large vesicles containing internal membrane, which form Ii-om early endosomal membranes at the cell periphery, are then transported on microtubules towards the pericentriolar region, and eventually dock onto and fuse with late endosomes. It is also becoming clear that the organization of early endosomes, and perhaps late endosomes, is very complex. Part of the early endosomes are formed by networks of thin tubules, which contain high amounts of recycling receptor molecules, but not the ligands destined to be degraded. The precise nature of these early endosomal membrane domains is unclear, however, as little is known about their molecular composition. Future studies will be required to understand the mechanisms controlling tubule biogenesis and internal membrane accumulation along the two axes of the pathway, recycling and transport to late endosomes, respectively. Major progress has been made in the past year in understanding some of the specialized routes of protein recycling from endosomes. Mellman, Pierre and Amigorena (pp 564-572) discuss in their review exciting observations from several groups which show that, in antigen-presenting cells, class II molecules of the MHC complex are loaded with antigenic peptides in specialized endosomal vesicles. These vesicles are
Editorial
presumably implicated in the recycling of class II molecules loaded with antigens to the cell surface. The relationships between these vesicles and traditional endosomes/lysosomes remain to be established. Class II vesicles can be distinguished l?om both early and late endosomes by several criteria, but reports differ on their lysosomal-like characteristics. A highly specialized recycling route from early endosomes occurs at the nerve terminal, which forms synaptic vesicles. The characterization of proteins implicated in synaptic vesicle targeting has been instrumental in formulating the SNARE (SNAP receptor) hypothesis. Bennett (pp 581-586) provides a detailed overview of the mechanisms controlling specificity of vesicle targeting and the role of SNARES. SNARES are proteins exposed on the cytoplasmic face of vesicles (v-SNARE) and target membranes (t-SNARE) which are believed to form a complex upon vesicle docking, and thereby mediate the specificity of vesicular transport [2]. SNARES interact with soluble components, SNAPS and the ATPase NSF, that are implicated in the formation of a stable complex released by ATP hydrolysis. Formation/dissociation of this complex has been suggested to power fusion of the vesicle and target membranes [2]. However, little is known on the mechanisms of intracellular membrane fusion. In addition to the SNARE-NSF-SNAP complex, other unidentifed proteins may be required to deform the bilayers and cause phospholipids to mix. Although progress has been made in unravelling the function of SNARE molecules and in studying their interactions, only a limited number of SNARES or SNARE-like proteins have been charactererized and localized in non-neuronal animal cells. Studies by Simons and co-workers in epithelial cells, in fact, suggest that biosynthetic membrane transport to the apical membrane domain may rely on a different mechanism utilizing annexin XIII [6]. Annexins have previously been implicated in membrane transport; in contrast to SNARES, they are not membrane proteins, but interact with the cytoplasmic face of many subcellular compartments [7]. Whether annexins and SNARES provide two fundamentally different mechanisms of membrane targeting/fusion or whether the role of both types of proteins is somehow interconnected is not known. Of particular interest in this context, is the discovery of a new protein (TAP/pl15), which is necessary for docking/fusion of both transcytotic vesicles to the apical membrane in liver cells and transport vesicles in intra-Golgi transport [8,9]. TAP/p1 15 may be a general factor mediating vesicle docking onto any target membrane, before the formation of the specific targeting complex. Proteins of the Rab family form a large family, whose members are essential for the regulation of targeting/fusion. These proteins have been the subject of extensive recent reviews (e.g. [lo]); however, the
overview
Gruenberg and Kreis
precise function of Rab proteins is still unclear. Recent studies from the groups of Rothman, Novick and Ferro-Novick suggest that Rab proteins, in fact, regulate or trigger the formation of SNARE complexes. It will clearly be important to identify and characterize the direct molecular targets of Rab proteins, in order to integrate their function in the complex process of membrane-membrane interactions. Finally, the role of cytoskeletal structures in cytoplasmic membrane traffic is essential and multifaceted. Microtubules play a key role in directing and facilitating membrane traffic in cells, and they are also implicated in the spatial arrangement of the organelles involved in membrane transport. Microtubule-based molecular motors, motor-activator complexes [ 1 l] and CLIPS, organelle-microtubule linker proteins [12], constitute the most important groups of factors regulating these processes. An extensive overview on this topic appeared in this year’s ‘Cytoskeleton’ issue of Current Opinion in Cell Biology [13] and the topic was thus not included here. However, as the family of unconventional myosins increases in members, evidence accumulates that actinbased processes may also significantly contribute toward regulation of membrane transport in cells (Hasson and Mooseker, pp 587-594). Intriguing, but so far not fully understood, observations indicate that the actin- and microtubule-based systems may play complementary roles in directing and regulating membrane traffic [14]. As is often the case in cell biology, new levels of complexity become apparent as new proteins are being discovered and as information becomes available on the biochemical properties of previously known components. The integration of all the information available on the molecular mechanisms of membrane transport is becoming increasingly complicated, partly because basic principles are still difficult to formulate and/or are quite speculative. Indeed, some of the fundamental properties of membranes remain obscure, including, for example, those controlling the bilayer fusion process. One of the major challenges in the years to come will clearly be to establish the hierarchy in the mode of action of known proteins, and to identify the components, including perhaps fundamental players, that are still missing.
References 1.
Krijnse Locker j, Ericsson M, Rottier PJ, Criffiths G: Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Colgi complex requires only one vesicular transport step. 1 Cell Biol 1994, 24:55-70.
2.
Rothman JE: Mechanisms of intracellular ture 1994, 376:55-63.
3.
Mellman I, Simons K: The Colgi complex: in vitro veritas? Ce// 1992, 68:829-840.
protein transport. I%-
521
522
Membranes and sortinrr 4.
Kreis TE, Lowe M, Pepperkok R: COPS regulating membrane traffic. Annu Rev Cell Dev Biol 1995, 11 :in press.
5.
Parton RG, Joggerst 6, Simons K: Regulated internalization caveolae. / Cell Biol 1994, 127:1199-l 215.
of
12.
Pierre P, Scheel J, Rickard JE, Kreis TE: CLIP-170 links endocytic vesicles to microtubules. Cell 1992, 70:887-900.
6.
Fiedler K, Lafont F, Parton RC, Simons K: Annexin Xlllb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. 1 Cell &o/1995, 128:1043-l 053.
13.
Cole NE%,Lippincott-Schwartz J: Organization of organelks and membrane traffic by microtubules. Curr Opin Cell Viol 1995, 7:55-64.
7.
Gruenberg J, Emans N: Annexins in membrane tranport. Cell Bio/ 1993, 3:224-227.
14.
Kuznetsov SA, Langford organelle movement in 356~722-725.
a.
nent of an activator of vesicle motility mediated by cytoplasmic dynein. / Cell Biol 1991, 115:1639-1650
Trends
Barrosi M, Nelson DS, Sztul E: Transcytosis-associated protein (TAP)/~~~s is a general fusion factor required for binding of vesicles to acceptor membranes. Pm Nat/ Acad Sci USA 1995, 92:527-531.
9.
Sapperstein SK, Walter DM, Crosvenor AR, Heuser JE, Waters MG: ~115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Colgi transport factor Usolp. Proc Nat1 Acad Sci USA 1995, 92:522-526.
10.
Pfeffer S: Rab CTPases: master regulators of membrane ficking. Curr Opin Cell Biol 1994, 6:522-526.
11.
Gill SR, Schroer TA, Szilak I, Steuer ER, Sheetz MP, Cleveland DW: Dynadin, a conserved, ubiquitously expressed compo-
traf-
GM, Weiss D: Actin-dependent squid axoplasm. Nature 1992,
J Gruenberg, Departement de Biochimie, Sciences II, 30 quai Ernest Ansermet, Switzerland. E-mail: [email protected]
UniversitP CH-1211,
de Genke, Ge&ve 4,
TE Kreis, Departement de Biologie Cellulaire, Universitk de GenPve, Sciences III, 30 quai Ernest Ansermet, CH-l?ll, Gentve 4, Switzerland. E-mail: [email protected]
Current
of Geneva,
Opinion
Geneva,
in Cell Biology
As the various events during the journey of a membrane or secretory protein from its site of synthesis, the endoplasmic reticulum (ER), to its final destination (e.g. the plasma membrane), continue to be unraveled, and ‘Membranes and sorting’ issues of Current Opinion in Cell Biology pass by, surprising findings are reported at regular intervals. Two of the most recent highlights of the past year’s discoveries indicate an additional unexpected function for the COPI coat proteins in retrieval of ER membrane proteins and the characterization of COPII, a third class of a transport vesicle associated coat essential for membrane traflic from the ER to the Golgi complex in yeast. The prospect that a family of coat proteins, related in structure and function, regulates all the different steps of intracellular membrane traffic appears to be becoming a reality The basis of this rapid pace of progress in understanding membrane biogenesis and cellular organization is founded on the combination of cellular genetics, molecular cell biology, biochemical reconstitution of defined processes in cell-free systems, and manipulation of living cells. We believe that a flavor of the power of this multidisciplinary approach is illustrated by the contributions in this issue on ‘Membranes and sorting’ by prominent scholars in the field. Helenius and his colleagues are deciphering the fine mechanisms of folding and maturation of membrane and secretory proteins (Hammond and Helenius, pp 523-529). An elaborate system of factors (including calnexin, calreticulin, ERGIC53/p58, BiP and other ER chaperones) is being unraveled, which ensures that only the correctly modified and properly folded proteins progress along the assembly line between the ER and Golgi complex and finally leave the early compartment(s) of biosynthetic membrane transport. Several of these factors are lectins that can discriminate between the different states of carbohydrate trimming on the transported proteins. In fact, a model is proposed in which these factors responsible for quality control build up an extended network or retention matrix that regulates exit from this first station of synthesis and transport to the next compartment.
Switzerland
1995,
7:519-522
ER proteins that have left their site of original function, for example because they are chaperoning newly synthesized protein to the next station in the biosynthetic pathway or because they have been missorted or escaped an ER retention mechanism, must be retrieved to ensure maximal efficiency of the system. Signals and some of the mechanisms that lead to retention or retrieval of proteins in the ER have been identified and characterized in detail (Pelham, pp 530-535). A recent exciting observation, also discussed in this article, implicates COPI, a coat associated with transport vesicles between the intermediate compartment and the Golgi complex, with retrieval of ER proteins containing the di-lysine ER-retention motif (KKXX in the single-letter code for amino acids; X is any amino acid). Biochemical evidence for direct interaction of polypeptides containing KKXX at their carboxyl terminus with COP1 has been obtained, and a genetical approach in yeast lead to the molecular characterization of U-COP (Retlp). The boundaries between the well defined rough ER and the cis-cisternae of the (medial) Golgi stack are so far only poorly defined. A compartment located between the ER and the Golgi complex has been proposed to exist and has been given many names (we use intermediate compartment in this overview). In addition, a network of tubular-cisternal structures, the cis-Golgi network, appears to be the port of entry for material destined to reach and transit through the cisternae of the Golgi complex. So far, the identity of both the intermediate compartment and the &Golgi network remains obscure, as no distinct membrane-bounded compartment has not been clearly defined between the rough ER and the stacked Golgi cisternae, presumably because of the highly dynamic nature of the membranes in this zone. In fact, continuity of ER membranes with the &most cisterna of the Golgi stack has recently been proposed by Gareth Grifiths and his colleagues [l]. It is essential to know the precise number and arrangement of the membrane-bounded compartments in the early biosynthetic membrane pathway, as these
Abbreviations ER-endoplasmic
reticulum; SNARE-SNAP
receptor; t-SNARE-target
SNARE; v-SNARE-vesicle
0 Current Biology Ltd ISSN 0955-0674
SNARE.
519
520
Membranes and sorting
parameters define the minimal number of vesicular steps required for transfer of cargo. So far, two classes of vesicle-associated coats implicated in membrane traffic between the ER and the Golgi complex have been characterized: COPI and, more recently, COP11 (Salama and Schekman, pp 536-543). The precise functions and steps of action of these two coat proteins are still enigmatic and thus different possible models are discussed. While COP11 appears to be involved exclusively in anterograde transport from the ER, the role of COPI may be more complex. Given the interaction of COP1 with some of the membrane proteins of the quality control machinery (i.e. proteins with KKXX ER-retention/retrieval signals), COP1 may be associated with shuttling of tranport intermediates or recycling the factors essential for assembly of membrane proteins until they are ready to reach the stacked cisternae of the Golgi complex. Transport through the cisternae of the medial Golgi has been studied and discussed in great detail [2]. Although several aspects in the current model describing membrane traffic through that organelle still need further clarification (for example, see [3,4]), reconstitution of this transport in vitro led to the discovery and molecular characterization of several ubiquitous key factors regulating membrane traffic. At the exit site of the Golgi complex, the trans-Golgi network, proteins are sorted into different vesicular carriers and delivered to their final destination. The example described in this issue addresses protein transport from the late Golgi, presumably the truns-Golgi network, to the yeast vacuole (Horazdovsky, DeWald and Emr, pp 544-551). Protein trafficking to the vacuole depends on a set of proteins whose homologues are also known to be implicated in other steps of vesicular transport, in yeast or mammalian cells. These include a small GTP-binding protein homologous to Rab5 (Vps2lp/Ypt5lp), a dynamin-like GTPase (Vpslp), and Seclp homologues (vps45p and Vps33p). Of particular interest is their finding that transport to the vacuole also depends on mositol 3-kinase (Vps34p). Membrane a phosphatid y 1’ association and activation of Vps34p appears to be mediated by a serine/threonine protein kinase, Vpsl5p. Several mechanisms by which vesicular traffic may be regulated via phosphoinositides are discussed. In their review, Lamaze and Schmid (pp 573-580) discuss the different routes of entry into animal cells. Receptor-mediated endocytosis via clathrin-coated pits and vesicles remains the best characterized endocytic pathway, and is probably the major route of plasma membrane protein and lipid turnover in most cell types. In vitro studies by Schmid and colleagues, together with the morphological studies of de Camilli and his group, now suggest that polymerization of the GTPase dynamin at the neck of an invaginated pit provides the molecular device which constricts the neck until it is severed from the membrane. Interestingly, uptake of solutes resumes after inhibition of the clathrin pathway
in a dynamin temperature-sensitive mutant, suggesting that an alternate pathway can compensate for the loss of the clathrin pathway. These observations agree with earlier observations indicating that an additional endocytic pathway leading to endosomes must exist. A third pathway, mediated by caveolae, has also been postulated. Studies carried out by Parton et al. [5] now unambiguously show that caveolae can detach from the cell surface, and form intracellular vesicles, albeit at a very low rate when compared to clathrin-coated vesicles. The intracellular destination of these vesicles, however, is still being debated. The cover of this issue illustrates the presence of a ‘bunch’ of these vesicles at the plasma membrane, emphasizing the intriguing complexity of the organization of caveolae in some cell types. The functional analogy between endocytosis in yeast and mammalian cells remains an important question. In yeast cells, studies from the groups of Riezman and Botstein indicate that endocytosis may be actin-driven, in contrast to clathrin-mediated internalization in mammalian cells. Homologues of molecules implicated in clathrin-mediated endocytosis in mammalian cells appear, in fact, to be necessary for vacuolar protein sorting in yeast (Rab5 and dynamin; see below and Horazdovsky, DeWald and Emr). Thus, Golgi-to-endosome transport in yeast has mechanistic similarities to clathrin-mediated internalization in mammalian cells. Gruenberg and Maxfield (pp 552-563) discuss membrane transport in the endocytic pathway. A comparison between in vivo and in vitro observations indicates that both early and late endosomes are highly dynamic organelles, connected by transport intermediates. These intermediates probably correspond to large vesicles containing internal membrane, which form Ii-om early endosomal membranes at the cell periphery, are then transported on microtubules towards the pericentriolar region, and eventually dock onto and fuse with late endosomes. It is also becoming clear that the organization of early endosomes, and perhaps late endosomes, is very complex. Part of the early endosomes are formed by networks of thin tubules, which contain high amounts of recycling receptor molecules, but not the ligands destined to be degraded. The precise nature of these early endosomal membrane domains is unclear, however, as little is known about their molecular composition. Future studies will be required to understand the mechanisms controlling tubule biogenesis and internal membrane accumulation along the two axes of the pathway, recycling and transport to late endosomes, respectively. Major progress has been made in the past year in understanding some of the specialized routes of protein recycling from endosomes. Mellman, Pierre and Amigorena (pp 564-572) discuss in their review exciting observations from several groups which show that, in antigen-presenting cells, class II molecules of the MHC complex are loaded with antigenic peptides in specialized endosomal vesicles. These vesicles are
Editorial
presumably implicated in the recycling of class II molecules loaded with antigens to the cell surface. The relationships between these vesicles and traditional endosomes/lysosomes remain to be established. Class II vesicles can be distinguished l?om both early and late endosomes by several criteria, but reports differ on their lysosomal-like characteristics. A highly specialized recycling route from early endosomes occurs at the nerve terminal, which forms synaptic vesicles. The characterization of proteins implicated in synaptic vesicle targeting has been instrumental in formulating the SNARE (SNAP receptor) hypothesis. Bennett (pp 581-586) provides a detailed overview of the mechanisms controlling specificity of vesicle targeting and the role of SNARES. SNARES are proteins exposed on the cytoplasmic face of vesicles (v-SNARE) and target membranes (t-SNARE) which are believed to form a complex upon vesicle docking, and thereby mediate the specificity of vesicular transport [2]. SNARES interact with soluble components, SNAPS and the ATPase NSF, that are implicated in the formation of a stable complex released by ATP hydrolysis. Formation/dissociation of this complex has been suggested to power fusion of the vesicle and target membranes [2]. However, little is known on the mechanisms of intracellular membrane fusion. In addition to the SNARE-NSF-SNAP complex, other unidentifed proteins may be required to deform the bilayers and cause phospholipids to mix. Although progress has been made in unravelling the function of SNARE molecules and in studying their interactions, only a limited number of SNARES or SNARE-like proteins have been charactererized and localized in non-neuronal animal cells. Studies by Simons and co-workers in epithelial cells, in fact, suggest that biosynthetic membrane transport to the apical membrane domain may rely on a different mechanism utilizing annexin XIII [6]. Annexins have previously been implicated in membrane transport; in contrast to SNARES, they are not membrane proteins, but interact with the cytoplasmic face of many subcellular compartments [7]. Whether annexins and SNARES provide two fundamentally different mechanisms of membrane targeting/fusion or whether the role of both types of proteins is somehow interconnected is not known. Of particular interest in this context, is the discovery of a new protein (TAP/pl15), which is necessary for docking/fusion of both transcytotic vesicles to the apical membrane in liver cells and transport vesicles in intra-Golgi transport [8,9]. TAP/p1 15 may be a general factor mediating vesicle docking onto any target membrane, before the formation of the specific targeting complex. Proteins of the Rab family form a large family, whose members are essential for the regulation of targeting/fusion. These proteins have been the subject of extensive recent reviews (e.g. [lo]); however, the
overview
Gruenberg and Kreis
precise function of Rab proteins is still unclear. Recent studies from the groups of Rothman, Novick and Ferro-Novick suggest that Rab proteins, in fact, regulate or trigger the formation of SNARE complexes. It will clearly be important to identify and characterize the direct molecular targets of Rab proteins, in order to integrate their function in the complex process of membrane-membrane interactions. Finally, the role of cytoskeletal structures in cytoplasmic membrane traffic is essential and multifaceted. Microtubules play a key role in directing and facilitating membrane traffic in cells, and they are also implicated in the spatial arrangement of the organelles involved in membrane transport. Microtubule-based molecular motors, motor-activator complexes [ 1 l] and CLIPS, organelle-microtubule linker proteins [12], constitute the most important groups of factors regulating these processes. An extensive overview on this topic appeared in this year’s ‘Cytoskeleton’ issue of Current Opinion in Cell Biology [13] and the topic was thus not included here. However, as the family of unconventional myosins increases in members, evidence accumulates that actinbased processes may also significantly contribute toward regulation of membrane transport in cells (Hasson and Mooseker, pp 587-594). Intriguing, but so far not fully understood, observations indicate that the actin- and microtubule-based systems may play complementary roles in directing and regulating membrane traffic [14]. As is often the case in cell biology, new levels of complexity become apparent as new proteins are being discovered and as information becomes available on the biochemical properties of previously known components. The integration of all the information available on the molecular mechanisms of membrane transport is becoming increasingly complicated, partly because basic principles are still difficult to formulate and/or are quite speculative. Indeed, some of the fundamental properties of membranes remain obscure, including, for example, those controlling the bilayer fusion process. One of the major challenges in the years to come will clearly be to establish the hierarchy in the mode of action of known proteins, and to identify the components, including perhaps fundamental players, that are still missing.
References 1.
Krijnse Locker j, Ericsson M, Rottier PJ, Criffiths G: Characterization of the budding compartment of mouse hepatitis virus: evidence that transport from the RER to the Colgi complex requires only one vesicular transport step. 1 Cell Biol 1994, 24:55-70.
2.
Rothman JE: Mechanisms of intracellular ture 1994, 376:55-63.
3.
Mellman I, Simons K: The Colgi complex: in vitro veritas? Ce// 1992, 68:829-840.
protein transport. I%-
521
522
Membranes and sortinrr 4.
Kreis TE, Lowe M, Pepperkok R: COPS regulating membrane traffic. Annu Rev Cell Dev Biol 1995, 11 :in press.
5.
Parton RG, Joggerst 6, Simons K: Regulated internalization caveolae. / Cell Biol 1994, 127:1199-l 215.
of
12.
Pierre P, Scheel J, Rickard JE, Kreis TE: CLIP-170 links endocytic vesicles to microtubules. Cell 1992, 70:887-900.
6.
Fiedler K, Lafont F, Parton RC, Simons K: Annexin Xlllb: a novel epithelial specific annexin is implicated in vesicular traffic to the apical plasma membrane. 1 Cell &o/1995, 128:1043-l 053.
13.
Cole NE%,Lippincott-Schwartz J: Organization of organelks and membrane traffic by microtubules. Curr Opin Cell Viol 1995, 7:55-64.
7.
Gruenberg J, Emans N: Annexins in membrane tranport. Cell Bio/ 1993, 3:224-227.
14.
Kuznetsov SA, Langford organelle movement in 356~722-725.
a.
nent of an activator of vesicle motility mediated by cytoplasmic dynein. / Cell Biol 1991, 115:1639-1650
Trends
Barrosi M, Nelson DS, Sztul E: Transcytosis-associated protein (TAP)/~~~s is a general fusion factor required for binding of vesicles to acceptor membranes. Pm Nat/ Acad Sci USA 1995, 92:527-531.
9.
Sapperstein SK, Walter DM, Crosvenor AR, Heuser JE, Waters MG: ~115 is a general vesicular transport factor related to the yeast endoplasmic reticulum to Colgi transport factor Usolp. Proc Nat1 Acad Sci USA 1995, 92:522-526.
10.
Pfeffer S: Rab CTPases: master regulators of membrane ficking. Curr Opin Cell Biol 1994, 6:522-526.
11.
Gill SR, Schroer TA, Szilak I, Steuer ER, Sheetz MP, Cleveland DW: Dynadin, a conserved, ubiquitously expressed compo-
traf-
GM, Weiss D: Actin-dependent squid axoplasm. Nature 1992,
J Gruenberg, Departement de Biochimie, Sciences II, 30 quai Ernest Ansermet, Switzerland. E-mail: [email protected]
UniversitP CH-1211,
de Genke, Ge&ve 4,
TE Kreis, Departement de Biologie Cellulaire, Universitk de GenPve, Sciences III, 30 quai Ernest Ansermet, CH-l?ll, Gentve 4, Switzerland. E-mail: [email protected]