- Email: [email protected]
8 complex
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Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex Shu-Chan Hsu, Christopher D. Hazuka, Davide L. Foletti and Richard H. Scheller The delivery of secretory vesicles to appropriate docking and fusion sites on the plasma membrane is crucial for many cellular functions, including formation of synapses, exocytosis of neurotransmitter, establishment and maintenance of cell polarity, cell growth and plasma membrane wound healing. Cell-biological, genetic and biochemical approaches have identified crucial proteins and protein interactions important for vesicle docking and fusion. However, a description of the molecular mechanisms underlying vesicle targeting to specific membrane-fusion sites remains elusive. This review discusses a set of proteins that might direct vesicles to specific domains of the plasma membrane.
have one additional subunit (exo84), and the 106-kDa band (p106) of the mammalian complex remains to be fully characterized. Mammalian p106 might be the homologue of the yeast Sec3p, but further characterization of the protein is required. Biochemical studies suggest the presence of one copy of each subunit per complex, which would yield a molecular mass of 753 kDa for the yeast complex and 734 kDa for the rat complex1,2. Similarities in the molecular masses of individual subunits and in the number of subunits in the complex in both species suggest that the yeast and rat complexes have a similar subunit organization and conformation. Visualization of the rat complex by quick-freeze, deep-etch electron microscopy showed that it adopts two classes of conformation depending on whether or not it has been fixed by glutaraldehyde2 (Fig. 1b). In the absence of glutaraldehyde fixation, the complex displayed a variety of forms, which generally comprised a central structure from which radiated a set of four to six arm-like structures. These arms ranged from 4–6 nm in width and 10–30 nm in length and often appeared to be split internally along their lengths. This extended conformation might be due to distortion or disruption of the unfixed complex upon its adsorption onto mica during preparation for electron microscopy. Fixation of the complex with glutaraldehyde prior to electron microscopy yielded a more reproducible and compact structure resembling the letter ‘T’or ‘Y’. The complex has a slightly elongated body, roughly the size of thyroglobulin, plus two arms that extend from one end of the body at varying angles through a flexible hinge region. Overall, the body of the complex measures 30 3 13 nm. For comparison, the diameter of a synaptic vesicle is approximately 50 nm. The arms of the fixed complex are approximately 15 3 6 nm, suggesting that they represent a subset of the shorter arms seen in unfixed samples. Likewise, the longer arms seen in the unfixed complex might pack together to form the body of the structure in the fixed samples. Although the functional significance of the structure is not yet clear, it will be interesting to map the organization of the subunits within this structure and study how distinct regions of this complex interact with other proteins to carry out its function.
The authors are in the Howard Hughes Medical Institute and the Dept of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, CA 94305, USA. E-mail: scheller@ cmgm.stanford. edu
Genetic and biochemical studies in budding yeast and mammalian systems have identified a protein complex, known as the exocyst, Sec3/5/6/8/10/15/ exo70 or sec6/8 complex (the ‘sec6/8 complex’ or the ‘complex’ for the purposes of this review), as an important component of the machinery that mediates exocytosis. This complex has been referred to as the sec6/8 complex because most of the work in mammalian systems has been performed using antibodies directed against the sec6 and sec8 subunits. In yeast, the complex (which is commonly referred to as the exocyst) comprises seven subunits, ranging from 70 to 144 kDa: Sec3p, -5p, -6p, -8p, -10p, -15p and exo701. In rat, the complex comprises eight subunits, ranging from 71 to 110 kDa (Ref. 2; Fig. 1a). Sequence analyses revealed that six subunits (Sec5, -6, -8, -10, -15 and exo70) of the yeast and rat complexes are homologous, with 21 to 24% sequence identity3–6. The mammalian complex appears to
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Roles of the sec6/8 complex Evidence in mammalian and yeast cells strongly indicates a role for the complex in the secretory pathway. In yeast, six subunits of the complex (Sec3, -5, -6, -8, -10 and -15) were originally identified as Sec proteins – mutations in the genes encoding these proteins inhibited secretion of invertase and resulted in the accumulation of secretory vesicles at the tip of the growing daughter cell, an area of intense exocytic activity7,8. During the yeast celldivision and budding processes, the complex is recruited to sites of rapid membrane growth9–12 (Fig. 2a). At the beginning of the budding process, the complex is concentrated at the tip of the growing daughter cell, where membrane addition takes place to support polarized growth of the daughter cell. Mutations in the gene encoding Sec3p result in trends in CELL BIOLOGY (Vol. 9) April 1999
reviews (a) random bud-site selection in diploid cells9,10,12, and expression of a truncated form of Sec10p containing only the final third of its sequence results in defective morphogenesis13. At a later stage in budding during cytokinesis, the complex is found at the mother– daughter cell boundary, where secretory vesicles fuse with the plasma membrane to form a septum between the mother and the daughter cell. These results suggest that the complex plays a role in directly targeting secretory vesicles to designated docking and fusion sites on the plasma membrane and/or in setting up targeting patches on the plasma membrane that receive vesicles at appropriate exocytic sites. It is interesting to note that the yeast complex, or at least the Sec3p subunit of this complex, might also be involved at earlier stages of the secretory pathway. Two mutant alleles of SEC3, sec3-4 and sec3-5, result in the accumulation of endoplasmic reticulum (ER) and Golgi complex membranes in addition to secretory vesicles, as well as in a partial block of carboxypeptidase Y transport from the ER to the Golgi complex and from the Golgi complex to the vacuole9. This result suggests that Sec3p functions, either by itself or as a member of the complex, in the intracellular vesicle-trafficking process in addition to the final stages of exocytosis. In mammals, the complex is expressed ubiquitously2–6,14. Mice possessing a mutation in the gene encoding Sec8 die early during embryogenesis15, suggesting that the complex is a universal component of the exocytic machinery in all cells. In the epithelial Madin–Darby canine kidney (MDCK) cell line, where Golgi-derived vesicles are targeted constantly to the plasma membrane, the complex is required continuously16 (Fig. 2b). Antibodies against rat Sec8 inhibit the delivery of low-density lipoprotein (LDL) receptors to the basolateral membrane, but not of p75 to the apical membrane, in polarized MDCK cells permeabilized with streptolysin-O. These results suggest that the complex plays a role in recruiting vesicles to specific domains on the plasma membrane. Interestingly, the intracellular localization of the complex in MDCK cells is dependent on cell polarity. In nonpolarized cells, the complex is found in a soluble form in the cytoplasm. After cell–cell contacts have been established, the complex localizes to the sites of cell–cell interactions along the plasma membrane. In fully polarized cells, the complex is further restricted in its localization to the plasma membrane at the apex of the basolateral domain (Fig. 2b). Its localization in polarized cells is similar to that of ZO-1, a protein associated with tight junctions. Upon disruption of E-cadherin-mediated cell–cell contact and cell polarity by EGTA, a Ca21 chelator, the complex dissociates from the plasma membrane. This cellpolarity-dependent recruitment of the complex suggests that it might play a role in establishing and/or maintaining polarized secretory pathways. In cultures of developing hippocampal neurons, sec6/8 immunoreactivity is present in the cell bodies, axons and in both dendritic and axonal growth cones17 (Fig. 2c). The presence of the complex in the growth cones is consistent with a function in membrane addition for growth cone expansion during trends in CELL BIOLOGY (Vol. 9) April 1999
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110 sec8 106 p106 102 sec5 96 sec15 86 sec6 84 exo84 79 exo70 71 sec10 734 kDa
FIGURE 1 The composition and structure of the sec6/8 complex. (a) Coomassie-blue-stained SDS polyacrylamide gel analysis of purified rat brain sec6/8 complex. The complex comprises eight proteins, ranging from 71–110 kDa. (b) Visualization of the sec6/8 complex structure by quick-freeze, deep-etch electron microscopy. Top row: structure of unfixed sec6/8 complex. The complex adopts a radially symmetric conformation in which there are arms of different lengths. Bottom two rows: structure of glutaraldehyde-fixed sec6/8 complex. The complex exhibits a compact structure resembling the letter ‘T’ or ‘Y’. It has a slightly elongated body whose dimensions are 30 3 13 nm, roughly the size of thyroglobulin, plus two arms that extend from one end of the body at varying angles. (Figure adapted, with permission, from Ref. 14.)
the development and remodelling of neural circuitry. In developing axons, but not dendrites, the complex clusters on the plasma membrane at periodic intervals of ~3.2 mm. Later in development, synaptic-vesicle markers colocalize with these complex clusters, suggesting an enrichment of Golgi-derived immature synaptic vesicles in those regions. Upon the formation of stable synapses, the sec6/8 immunoreactivity disappears, leaving behind a cluster of mature synaptic vesicles. These observations suggest that the complex plays a role in recruiting and/or retaining synaptic vesicles at potential active zones during synaptogenesis but is not required for local cycling of mature synaptic vesicles in established synapses. Molecular basis of sec6/8 complex function A common role for the complex in the aforementioned diversity of cellular processes is the site-specific recruitment of secretory vesicles to designated exocytic sites on the plasma membrane. What are the molecular mechanisms of its function? It is interesting to note that, although the amino acid sequences of subunits of the complex predict soluble proteins, ~40% of the yeast complex18 and 70–90% of the mammalian complex is found associated with membranes, most likely the plasma membrane2,16. It remains to be determined whether both the cytosolic and membrane-bound pools of the complex are functionally active or whether the cytosolic fraction is simply a reservoir for membrane recruitment when the complex is needed at particular domains of the plasma membrane. In either case, the focal localization of the complex at specific membrane sites in response to cellular cues is probably due to relocation of the existing
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FIGURE 2 A model of the actions of the sec6/8 complex in vesicle accumulation and synaptogenesis. (a) Actions of the sec6/8 complex during the life cycle of the budding yeast. The sec6/8 complex (shown in orange) is present at sites of secretion in the tip of the budding daughter cell, at sites of cytokinesis in dividing cells and in a patch at the bud scar that designates the site of exocytosis underlying future bud formation [there is a cytosolic pool as well (orange dots)]8. (b) A model of the role of the sec6/8 complex in polarized secretion in Madin–Darby canine kidney (MDCK) epithelial cells. Unpolarized MDCK cells target vesicles (shown in green and red) to random areas of the plasma membrane. The sec6/8 complex (orange) is dispersed throughout the cytosol. Upon the addition of Ca21, two cells contact each other and the cytoplasmic sec6/8 complex is reorganized to the contacting plasma membrane. Once polarized, the cells target vesicles either to the apical membrane (green) or the basolateral membrane (red). The sec6/8 complex is located along the membrane near the tight junctions, suggesting that the junction is a site to which vesicles are targeted. Upon depletion of Ca21, the cells become unpolarized and the sec6/8 complex disperses to the cytosol16. (c) A model of the role of the sec6/8 complex in synaptogenesis. Neurons of increasing maturity are shown from left to right. In young neurons, the sec6/8 complex (orange) is present in growth cones of neurites. Later, one neurite becomes the axon, while the others develop into dendrites; the sec6/8 complex is organized into periodic domains along the axon. As the neuron matures, vesicle clusters are found in some of the sec6/8 domains (synapsin-1-containing vesicle clusters are shown in blue). As synapses are formed between the axon and dendrites (a dendrite is shown in grey), local clusters of synaptic vesicles are stabilized and the sec6/8 complex is downregulated. Abbreviation: N, nucleus.
complex from the cytoplasm rather than recruitment of newly synthesized complex to those sites16. Possible mechanisms for the recruitment of the complex to specific domains of the plasma membrane include posttranslational modifications such as phosphorylation and/or fatty acylation, interaction with transient signal-transduction messenger molecules, modification of its putative membrane receptors, transient expression of new membrane receptors, modification of cytoskeletal interactions or a combination of these mechanisms. It is not clear whether the relocation of the sec6/8 complex requires rearrangement of cytoskeletal elements. Genetic studies in yeast suggest an interdependence between Sec3p and profilin (an actin-binding protein)9,10, and between Sec6p and
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actin/myo2 (a type V myosin important for secretion). Dissociation of the actin network by an actin inhibitor (latrunculin-A) resulted in the mislocalization of Sec8p. These results suggest that the recruitment of the sec6/8 complex to exocytic sites requires either a direct or an indirect interaction with an actinmediated trafficking pathway19. Localization studies using Sec3p tagged with green fluorescent protein (Sec3–GFP), however, showed that the recruitment of Sec3–GFP to the plasma membrane persists after the dissociation of the actin network by latrunculin-A12. In fact, the Sec3–GFP localization is independent of secretory vesicle trafficking, suggesting a role in marking potential exocytic sites prior to the recruitment of secretory vesicles to the plasma membrane. It is possible that Sec3–GFP exists in more than one state: as a trends in CELL BIOLOGY (Vol. 9) April 1999
reviews component of the complex, by itself or in association with other as-yet-unidentified proteins. When Sec3–GFP is not part of the complex, it might be recruited to the membrane in an actin-independent manner to mark potential exocytic sites; when Sec3–GFP is associated with the complex, it might participate in vesicle targeting in coordination with actin and other cytoskeletal elements. However, it is not yet clear whether individual mammalian sec6/8 complex subunits can function independently. Monitoring the organization of individual subunits with subunit-specific antibodies and/or GFP-tagged subunits will shed light on the potential roles of the various assembly states of the complex. Taken together, the data from yeast and mammalian cells suggest that the complex plays a role in targeting vesicles to specific domains on the plasma membrane, possibly by setting up or maintaining a plasma membrane ‘targeting patch’ that serves to recruit or receive vesicles20. However, the subunits of the complex do not contain previously characterized motifs, making it difficult to form hypotheses about the mechanisms of their function. One way to overcome this limitation is to identify and characterize proteins that might interact with the complex in the cytoplasm, at the plasma membrane and on vesicles. Changes in protein interactions that depend on various cellular states such as polarization, neuronal development and synaptogenesis can also be studied by immunoprecipitation. For example, by using monoclonal antibodies directed against rat Sec8, it has already been shown that the cytosolic rat brain complex co-immunoprecipitates with four septins14: KIAA0128, CDC10, NEDD5 and H521–24. The septin proteins comprise a novel class of cytoskeletal elements. Similar to members of the sec6/8 complex, the septins are important in a plethora of cellular processes that involve either membrane and protein addition to the plasma membrane or generation of cell polarity25–31. These studies suggest that the sec6/8 and septin complexes serve as a transient focal scaffold for recruiting and targeting proteins and vesicles to appropriate domains on the plasma membrane during different stages of the cell cycle and development. Furthermore, the large size of the sec6/8 complex suggests that it might interact with many proteins simultaneously; potential candidates include cytoskeletal elements and their regulators, other cytosolic constituents in the vesicle targeting/ docking/fusion pathway and plasma membrane/ secretory-vesicle proteins. Thus, it might be possible in the future to use the sec6/8 complex as a molecular handle to further define other components of the vesicle-trafficking process, particularly those important in targeting mechanisms. A link between signal transduction and vesicle trafficking? Vesicle-trafficking pathways are regulated dynamically during the cell cycle and development, although communication between the vesicletrafficking process and the wide variety of temporal and spatial cellular cues is not well understood. To ensure that secretory vesicles are delivered to the trends in CELL BIOLOGY (Vol. 9) April 1999
correct location at the appropriate time, the vesicletrafficking machinery must be responsive to signaltransduction pathways. Presently, the molecular mechanisms of such communication are unknown, although it is attractive to speculate that, in response to cellular cues, the plasma membrane vesicletargeting sites could be changed spatially or modified functionally. Modifications of the sec6/8 complex, the cytoskeletal network and cytoskeleton-interacting proteins are likely to be important in regulating delivery of vesicles to their designated exocytic sites, so that the docking and fusion machinery can negotiate the exocytosis event. The identification and characterization of proteins and protein interactions mediating and regulating these events will not only further our understanding of vesicle trafficking but will also provide a foundation for future experimentation to study how regulation of vesicle trafficking can modulate physiological functions. References 1 TerBush, D. R. et al. (1996) EMBO J. 15, 6483–6494 2 Hsu, S. C. et al. (1996) Neuron 17, 1209–1219 3 Ting, A. E. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9613–9617 4 Hazuka, C. D., Hsu, S-C. and Scheller, R. H. (1997) Gene 187, 67–73 5 Guo, W. et al. (1997) FEBS Lett. 404, 135–139 6 Kee, Y. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14438–14443 7 Novick, P., Field, C. and Schekman, R. (1980) Cell 21, 205–215 8 Finger, F. P. and Novick, P. (1998) J. Cell Biol. 142, 609–612 9 Finger, F. P. and Novick, P. (1997) Mol. Biol. Cell 8, 647–662 10 Haarer, B. K. et al. (1996) Genetics 144, 495–510 11 Mondésert, G., Clarke, D. J. and Reed, S. I. (1997) Genetics 147, 421–434 12 Finger, F. P., Hughes, T. E. and Novick, P. (1998) Cell 92, 559–571 13 Roth, D., Guo, W. and Novick, P. (1998) Mol. Biol. Cell 9, 1725–1739 14 Hsu, S-C. et al. (1998) Neuron 20, 1111–1122 15 Friedrich, G. A., Hildebrand, J. D. and Soriano, P. (1997) Dev. Biol. 192, 364–374 16 Grindstaff, K. K. et al. (1998) Cell 93, 731–740 17 Hazuka, C. D. et al. (1999) J. Neurosci. 19, 1324–1334 18 Bowser, R. et al. (1992) J. Cell Biol. 118, 1041–1056 19 Ayscough, K. R. et al. (1997) J. Cell Biol. 137, 399–416 20 Drubin, D. G. and Nelson, W. J. (1996) Cell 84, 335–344 21 Nagase, T. et al. (1995) DNA Res. 2, 167–174 22 Nakatsuru, S., Sudo, K. and Nakamura, Y. (1994) Biochem. Biophys. Res. Commun. 202, 82–87 23 Kumar, S., Tomooka, Y. and Noda, M. (1992) Biochem. Biophys. Res. Commun. 185, 1155–1161 24 Kinoshita, M. et al. (1997) Genes Dev. 11, 1535–1547 25 Flescher, E. G., Madden, K. and Snyder, M. (1993) J. Cell Biol. 122, 373–386 26 Neufeld, T. P. and Rubin, G. M. (1994) Cell 77, 371–379 27 Fares, H., Peifer, M. and Pringle, J. R. (1995) Mol. Biol. Cell 6, 1843–1859 28 Chant, J. et al. (1995) J. Cell Biol. 129, 767–778 29 Konopka, J. B., DeMattei, C. and Davis, C. (1995) Mol. Cell Biol. 15, 723–730 30 Fares, H., Goetsch, L. and Pringle, J. R. (1996) J. Cell Biol. 132, 399–411 31 Longtine, M. S. et al. (1996) Curr. Opin. Cell Biol. 8, 106–119
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Targeting vesicles to specific sites on the plasma membrane: the role of the sec6/8 complex Shu-Chan Hsu, Christopher D. Hazuka, Davide L. Foletti and Richard H. Scheller The delivery of secretory vesicles to appropriate docking and fusion sites on the plasma membrane is crucial for many cellular functions, including formation of synapses, exocytosis of neurotransmitter, establishment and maintenance of cell polarity, cell growth and plasma membrane wound healing. Cell-biological, genetic and biochemical approaches have identified crucial proteins and protein interactions important for vesicle docking and fusion. However, a description of the molecular mechanisms underlying vesicle targeting to specific membrane-fusion sites remains elusive. This review discusses a set of proteins that might direct vesicles to specific domains of the plasma membrane.
have one additional subunit (exo84), and the 106-kDa band (p106) of the mammalian complex remains to be fully characterized. Mammalian p106 might be the homologue of the yeast Sec3p, but further characterization of the protein is required. Biochemical studies suggest the presence of one copy of each subunit per complex, which would yield a molecular mass of 753 kDa for the yeast complex and 734 kDa for the rat complex1,2. Similarities in the molecular masses of individual subunits and in the number of subunits in the complex in both species suggest that the yeast and rat complexes have a similar subunit organization and conformation. Visualization of the rat complex by quick-freeze, deep-etch electron microscopy showed that it adopts two classes of conformation depending on whether or not it has been fixed by glutaraldehyde2 (Fig. 1b). In the absence of glutaraldehyde fixation, the complex displayed a variety of forms, which generally comprised a central structure from which radiated a set of four to six arm-like structures. These arms ranged from 4–6 nm in width and 10–30 nm in length and often appeared to be split internally along their lengths. This extended conformation might be due to distortion or disruption of the unfixed complex upon its adsorption onto mica during preparation for electron microscopy. Fixation of the complex with glutaraldehyde prior to electron microscopy yielded a more reproducible and compact structure resembling the letter ‘T’or ‘Y’. The complex has a slightly elongated body, roughly the size of thyroglobulin, plus two arms that extend from one end of the body at varying angles through a flexible hinge region. Overall, the body of the complex measures 30 3 13 nm. For comparison, the diameter of a synaptic vesicle is approximately 50 nm. The arms of the fixed complex are approximately 15 3 6 nm, suggesting that they represent a subset of the shorter arms seen in unfixed samples. Likewise, the longer arms seen in the unfixed complex might pack together to form the body of the structure in the fixed samples. Although the functional significance of the structure is not yet clear, it will be interesting to map the organization of the subunits within this structure and study how distinct regions of this complex interact with other proteins to carry out its function.
The authors are in the Howard Hughes Medical Institute and the Dept of Molecular and Cellular Physiology, Stanford University Medical School, Stanford, CA 94305, USA. E-mail: scheller@ cmgm.stanford. edu
Genetic and biochemical studies in budding yeast and mammalian systems have identified a protein complex, known as the exocyst, Sec3/5/6/8/10/15/ exo70 or sec6/8 complex (the ‘sec6/8 complex’ or the ‘complex’ for the purposes of this review), as an important component of the machinery that mediates exocytosis. This complex has been referred to as the sec6/8 complex because most of the work in mammalian systems has been performed using antibodies directed against the sec6 and sec8 subunits. In yeast, the complex (which is commonly referred to as the exocyst) comprises seven subunits, ranging from 70 to 144 kDa: Sec3p, -5p, -6p, -8p, -10p, -15p and exo701. In rat, the complex comprises eight subunits, ranging from 71 to 110 kDa (Ref. 2; Fig. 1a). Sequence analyses revealed that six subunits (Sec5, -6, -8, -10, -15 and exo70) of the yeast and rat complexes are homologous, with 21 to 24% sequence identity3–6. The mammalian complex appears to
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Roles of the sec6/8 complex Evidence in mammalian and yeast cells strongly indicates a role for the complex in the secretory pathway. In yeast, six subunits of the complex (Sec3, -5, -6, -8, -10 and -15) were originally identified as Sec proteins – mutations in the genes encoding these proteins inhibited secretion of invertase and resulted in the accumulation of secretory vesicles at the tip of the growing daughter cell, an area of intense exocytic activity7,8. During the yeast celldivision and budding processes, the complex is recruited to sites of rapid membrane growth9–12 (Fig. 2a). At the beginning of the budding process, the complex is concentrated at the tip of the growing daughter cell, where membrane addition takes place to support polarized growth of the daughter cell. Mutations in the gene encoding Sec3p result in trends in CELL BIOLOGY (Vol. 9) April 1999
reviews (a) random bud-site selection in diploid cells9,10,12, and expression of a truncated form of Sec10p containing only the final third of its sequence results in defective morphogenesis13. At a later stage in budding during cytokinesis, the complex is found at the mother– daughter cell boundary, where secretory vesicles fuse with the plasma membrane to form a septum between the mother and the daughter cell. These results suggest that the complex plays a role in directly targeting secretory vesicles to designated docking and fusion sites on the plasma membrane and/or in setting up targeting patches on the plasma membrane that receive vesicles at appropriate exocytic sites. It is interesting to note that the yeast complex, or at least the Sec3p subunit of this complex, might also be involved at earlier stages of the secretory pathway. Two mutant alleles of SEC3, sec3-4 and sec3-5, result in the accumulation of endoplasmic reticulum (ER) and Golgi complex membranes in addition to secretory vesicles, as well as in a partial block of carboxypeptidase Y transport from the ER to the Golgi complex and from the Golgi complex to the vacuole9. This result suggests that Sec3p functions, either by itself or as a member of the complex, in the intracellular vesicle-trafficking process in addition to the final stages of exocytosis. In mammals, the complex is expressed ubiquitously2–6,14. Mice possessing a mutation in the gene encoding Sec8 die early during embryogenesis15, suggesting that the complex is a universal component of the exocytic machinery in all cells. In the epithelial Madin–Darby canine kidney (MDCK) cell line, where Golgi-derived vesicles are targeted constantly to the plasma membrane, the complex is required continuously16 (Fig. 2b). Antibodies against rat Sec8 inhibit the delivery of low-density lipoprotein (LDL) receptors to the basolateral membrane, but not of p75 to the apical membrane, in polarized MDCK cells permeabilized with streptolysin-O. These results suggest that the complex plays a role in recruiting vesicles to specific domains on the plasma membrane. Interestingly, the intracellular localization of the complex in MDCK cells is dependent on cell polarity. In nonpolarized cells, the complex is found in a soluble form in the cytoplasm. After cell–cell contacts have been established, the complex localizes to the sites of cell–cell interactions along the plasma membrane. In fully polarized cells, the complex is further restricted in its localization to the plasma membrane at the apex of the basolateral domain (Fig. 2b). Its localization in polarized cells is similar to that of ZO-1, a protein associated with tight junctions. Upon disruption of E-cadherin-mediated cell–cell contact and cell polarity by EGTA, a Ca21 chelator, the complex dissociates from the plasma membrane. This cellpolarity-dependent recruitment of the complex suggests that it might play a role in establishing and/or maintaining polarized secretory pathways. In cultures of developing hippocampal neurons, sec6/8 immunoreactivity is present in the cell bodies, axons and in both dendritic and axonal growth cones17 (Fig. 2c). The presence of the complex in the growth cones is consistent with a function in membrane addition for growth cone expansion during trends in CELL BIOLOGY (Vol. 9) April 1999
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110 sec8 106 p106 102 sec5 96 sec15 86 sec6 84 exo84 79 exo70 71 sec10 734 kDa
FIGURE 1 The composition and structure of the sec6/8 complex. (a) Coomassie-blue-stained SDS polyacrylamide gel analysis of purified rat brain sec6/8 complex. The complex comprises eight proteins, ranging from 71–110 kDa. (b) Visualization of the sec6/8 complex structure by quick-freeze, deep-etch electron microscopy. Top row: structure of unfixed sec6/8 complex. The complex adopts a radially symmetric conformation in which there are arms of different lengths. Bottom two rows: structure of glutaraldehyde-fixed sec6/8 complex. The complex exhibits a compact structure resembling the letter ‘T’ or ‘Y’. It has a slightly elongated body whose dimensions are 30 3 13 nm, roughly the size of thyroglobulin, plus two arms that extend from one end of the body at varying angles. (Figure adapted, with permission, from Ref. 14.)
the development and remodelling of neural circuitry. In developing axons, but not dendrites, the complex clusters on the plasma membrane at periodic intervals of ~3.2 mm. Later in development, synaptic-vesicle markers colocalize with these complex clusters, suggesting an enrichment of Golgi-derived immature synaptic vesicles in those regions. Upon the formation of stable synapses, the sec6/8 immunoreactivity disappears, leaving behind a cluster of mature synaptic vesicles. These observations suggest that the complex plays a role in recruiting and/or retaining synaptic vesicles at potential active zones during synaptogenesis but is not required for local cycling of mature synaptic vesicles in established synapses. Molecular basis of sec6/8 complex function A common role for the complex in the aforementioned diversity of cellular processes is the site-specific recruitment of secretory vesicles to designated exocytic sites on the plasma membrane. What are the molecular mechanisms of its function? It is interesting to note that, although the amino acid sequences of subunits of the complex predict soluble proteins, ~40% of the yeast complex18 and 70–90% of the mammalian complex is found associated with membranes, most likely the plasma membrane2,16. It remains to be determined whether both the cytosolic and membrane-bound pools of the complex are functionally active or whether the cytosolic fraction is simply a reservoir for membrane recruitment when the complex is needed at particular domains of the plasma membrane. In either case, the focal localization of the complex at specific membrane sites in response to cellular cues is probably due to relocation of the existing
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FIGURE 2 A model of the actions of the sec6/8 complex in vesicle accumulation and synaptogenesis. (a) Actions of the sec6/8 complex during the life cycle of the budding yeast. The sec6/8 complex (shown in orange) is present at sites of secretion in the tip of the budding daughter cell, at sites of cytokinesis in dividing cells and in a patch at the bud scar that designates the site of exocytosis underlying future bud formation [there is a cytosolic pool as well (orange dots)]8. (b) A model of the role of the sec6/8 complex in polarized secretion in Madin–Darby canine kidney (MDCK) epithelial cells. Unpolarized MDCK cells target vesicles (shown in green and red) to random areas of the plasma membrane. The sec6/8 complex (orange) is dispersed throughout the cytosol. Upon the addition of Ca21, two cells contact each other and the cytoplasmic sec6/8 complex is reorganized to the contacting plasma membrane. Once polarized, the cells target vesicles either to the apical membrane (green) or the basolateral membrane (red). The sec6/8 complex is located along the membrane near the tight junctions, suggesting that the junction is a site to which vesicles are targeted. Upon depletion of Ca21, the cells become unpolarized and the sec6/8 complex disperses to the cytosol16. (c) A model of the role of the sec6/8 complex in synaptogenesis. Neurons of increasing maturity are shown from left to right. In young neurons, the sec6/8 complex (orange) is present in growth cones of neurites. Later, one neurite becomes the axon, while the others develop into dendrites; the sec6/8 complex is organized into periodic domains along the axon. As the neuron matures, vesicle clusters are found in some of the sec6/8 domains (synapsin-1-containing vesicle clusters are shown in blue). As synapses are formed between the axon and dendrites (a dendrite is shown in grey), local clusters of synaptic vesicles are stabilized and the sec6/8 complex is downregulated. Abbreviation: N, nucleus.
complex from the cytoplasm rather than recruitment of newly synthesized complex to those sites16. Possible mechanisms for the recruitment of the complex to specific domains of the plasma membrane include posttranslational modifications such as phosphorylation and/or fatty acylation, interaction with transient signal-transduction messenger molecules, modification of its putative membrane receptors, transient expression of new membrane receptors, modification of cytoskeletal interactions or a combination of these mechanisms. It is not clear whether the relocation of the sec6/8 complex requires rearrangement of cytoskeletal elements. Genetic studies in yeast suggest an interdependence between Sec3p and profilin (an actin-binding protein)9,10, and between Sec6p and
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actin/myo2 (a type V myosin important for secretion). Dissociation of the actin network by an actin inhibitor (latrunculin-A) resulted in the mislocalization of Sec8p. These results suggest that the recruitment of the sec6/8 complex to exocytic sites requires either a direct or an indirect interaction with an actinmediated trafficking pathway19. Localization studies using Sec3p tagged with green fluorescent protein (Sec3–GFP), however, showed that the recruitment of Sec3–GFP to the plasma membrane persists after the dissociation of the actin network by latrunculin-A12. In fact, the Sec3–GFP localization is independent of secretory vesicle trafficking, suggesting a role in marking potential exocytic sites prior to the recruitment of secretory vesicles to the plasma membrane. It is possible that Sec3–GFP exists in more than one state: as a trends in CELL BIOLOGY (Vol. 9) April 1999
reviews component of the complex, by itself or in association with other as-yet-unidentified proteins. When Sec3–GFP is not part of the complex, it might be recruited to the membrane in an actin-independent manner to mark potential exocytic sites; when Sec3–GFP is associated with the complex, it might participate in vesicle targeting in coordination with actin and other cytoskeletal elements. However, it is not yet clear whether individual mammalian sec6/8 complex subunits can function independently. Monitoring the organization of individual subunits with subunit-specific antibodies and/or GFP-tagged subunits will shed light on the potential roles of the various assembly states of the complex. Taken together, the data from yeast and mammalian cells suggest that the complex plays a role in targeting vesicles to specific domains on the plasma membrane, possibly by setting up or maintaining a plasma membrane ‘targeting patch’ that serves to recruit or receive vesicles20. However, the subunits of the complex do not contain previously characterized motifs, making it difficult to form hypotheses about the mechanisms of their function. One way to overcome this limitation is to identify and characterize proteins that might interact with the complex in the cytoplasm, at the plasma membrane and on vesicles. Changes in protein interactions that depend on various cellular states such as polarization, neuronal development and synaptogenesis can also be studied by immunoprecipitation. For example, by using monoclonal antibodies directed against rat Sec8, it has already been shown that the cytosolic rat brain complex co-immunoprecipitates with four septins14: KIAA0128, CDC10, NEDD5 and H521–24. The septin proteins comprise a novel class of cytoskeletal elements. Similar to members of the sec6/8 complex, the septins are important in a plethora of cellular processes that involve either membrane and protein addition to the plasma membrane or generation of cell polarity25–31. These studies suggest that the sec6/8 and septin complexes serve as a transient focal scaffold for recruiting and targeting proteins and vesicles to appropriate domains on the plasma membrane during different stages of the cell cycle and development. Furthermore, the large size of the sec6/8 complex suggests that it might interact with many proteins simultaneously; potential candidates include cytoskeletal elements and their regulators, other cytosolic constituents in the vesicle targeting/ docking/fusion pathway and plasma membrane/ secretory-vesicle proteins. Thus, it might be possible in the future to use the sec6/8 complex as a molecular handle to further define other components of the vesicle-trafficking process, particularly those important in targeting mechanisms. A link between signal transduction and vesicle trafficking? Vesicle-trafficking pathways are regulated dynamically during the cell cycle and development, although communication between the vesicletrafficking process and the wide variety of temporal and spatial cellular cues is not well understood. To ensure that secretory vesicles are delivered to the trends in CELL BIOLOGY (Vol. 9) April 1999
correct location at the appropriate time, the vesicletrafficking machinery must be responsive to signaltransduction pathways. Presently, the molecular mechanisms of such communication are unknown, although it is attractive to speculate that, in response to cellular cues, the plasma membrane vesicletargeting sites could be changed spatially or modified functionally. Modifications of the sec6/8 complex, the cytoskeletal network and cytoskeleton-interacting proteins are likely to be important in regulating delivery of vesicles to their designated exocytic sites, so that the docking and fusion machinery can negotiate the exocytosis event. The identification and characterization of proteins and protein interactions mediating and regulating these events will not only further our understanding of vesicle trafficking but will also provide a foundation for future experimentation to study how regulation of vesicle trafficking can modulate physiological functions. References 1 TerBush, D. R. et al. (1996) EMBO J. 15, 6483–6494 2 Hsu, S. C. et al. (1996) Neuron 17, 1209–1219 3 Ting, A. E. et al. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 9613–9617 4 Hazuka, C. D., Hsu, S-C. and Scheller, R. H. (1997) Gene 187, 67–73 5 Guo, W. et al. (1997) FEBS Lett. 404, 135–139 6 Kee, Y. et al. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 14438–14443 7 Novick, P., Field, C. and Schekman, R. (1980) Cell 21, 205–215 8 Finger, F. P. and Novick, P. (1998) J. Cell Biol. 142, 609–612 9 Finger, F. P. and Novick, P. (1997) Mol. Biol. Cell 8, 647–662 10 Haarer, B. K. et al. (1996) Genetics 144, 495–510 11 Mondésert, G., Clarke, D. J. and Reed, S. I. (1997) Genetics 147, 421–434 12 Finger, F. P., Hughes, T. E. and Novick, P. (1998) Cell 92, 559–571 13 Roth, D., Guo, W. and Novick, P. (1998) Mol. Biol. Cell 9, 1725–1739 14 Hsu, S-C. et al. (1998) Neuron 20, 1111–1122 15 Friedrich, G. A., Hildebrand, J. D. and Soriano, P. (1997) Dev. Biol. 192, 364–374 16 Grindstaff, K. K. et al. (1998) Cell 93, 731–740 17 Hazuka, C. D. et al. (1999) J. Neurosci. 19, 1324–1334 18 Bowser, R. et al. (1992) J. Cell Biol. 118, 1041–1056 19 Ayscough, K. R. et al. (1997) J. Cell Biol. 137, 399–416 20 Drubin, D. G. and Nelson, W. J. (1996) Cell 84, 335–344 21 Nagase, T. et al. (1995) DNA Res. 2, 167–174 22 Nakatsuru, S., Sudo, K. and Nakamura, Y. (1994) Biochem. Biophys. Res. Commun. 202, 82–87 23 Kumar, S., Tomooka, Y. and Noda, M. (1992) Biochem. Biophys. Res. Commun. 185, 1155–1161 24 Kinoshita, M. et al. (1997) Genes Dev. 11, 1535–1547 25 Flescher, E. G., Madden, K. and Snyder, M. (1993) J. Cell Biol. 122, 373–386 26 Neufeld, T. P. and Rubin, G. M. (1994) Cell 77, 371–379 27 Fares, H., Peifer, M. and Pringle, J. R. (1995) Mol. Biol. Cell 6, 1843–1859 28 Chant, J. et al. (1995) J. Cell Biol. 129, 767–778 29 Konopka, J. B., DeMattei, C. and Davis, C. (1995) Mol. Cell Biol. 15, 723–730 30 Fares, H., Goetsch, L. and Pringle, J. R. (1996) J. Cell Biol. 132, 399–411 31 Longtine, M. S. et al. (1996) Curr. Opin. Cell Biol. 8, 106–119
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