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52 Cytoskeletal proteins and Golgi dynamics Jennifer Lippincott-Schwartz Association of the Golgi complex with cytoskeletal elements, in particular microtubules, is required for maintenance of the Golgi's characteristic spatial location within cells and for efficient delivery of proteins and lipids to diverse cellular sites. Recent work has suggested the mechanisms underlying this association involve components, such as ankyrin and spectrin, that facilitate Golgi membrane association with motor proteins, including cytoplasmic dynein, kinesin and myosin. Understanding how these associations are regulated and what roles they play in Golgi trafficking and dynamics is fundamental for insight into the spatial and functional integration of secretory membrane traffic. such cells usually emanate from a perinuclear microtubule- organizing-center (MTOC), forming a uniformly polarized radial array--minus ends of microtubules converging at the cell center and plus ends scattered in the cell periphery. The Golgi complex is invariably centered around the MTOC (Figure 1) and actively maintained there [5,6,7]. This localization is ideal for enabling cargo-enriched transport intermediates derived from the ER to use the radially arranged microtubules to converge at one central location. Secretory cargo can then be processed, and repackaged into post-Golgi carriers which move out again along microtubule tracks to the cell surface. Addresses Cell Biology and Metabolism Branch, NICHD, National Institute for Health, Bethesda, MD 20892-0001, USA; e-mail: jlippin@helix.nih.gov Current Opinion in Cell Biology 1998, 10:59-59 http:l/biomednet.comlelecref/0955067401000052 © Current Biology Ltd ISSN 0955-0674 Abbreviations ARF ADP-ribosylation factor BFA brefeldin A COPI coatomer DHC2 dynein heavy chain 2 ER endoplasmic reticulum GFP green fluorescent protein MTOC microtubule-organizing-center TGN trans Golgi network VSVG vesicular stornatitis viral G protein In t roduct ion The Golgi complex is a dynamic, steady-state membrane system in close communication with the endoplasmic reticulum (ER), and plays a central role in protein and lipid processing and trafficking within cells [1,2]. It receives newly synthesized molecules from the ER, covalently modifies them and then distributes them to the plasma membrane, lysosomes and secretory vesicles. The Golgi also acts as a countercurrent fractionation system to recycle components that have entered the secretory pathway back to the ER. These diverse roles are dependent on small membrane-bound structures that continuously shuttle membrane into and out of the Golgi's polarized stack of enzyme-enriched, flattened cisternae. By regulating membrane flux into and out of the Golgi complex, these transport intermediates control the steady-state size and location of the Golgi complex and ensure its proper functioning within cells. In higher eukaryotic cells, Golgi-targeted/derived transport intermediates use microtubules and their associated motor proteins to move through the cytoplasm [3]. Free diffusion is simply too slow and haphazard [4]. Microtubules in A major focus of current work in this area has been clarifi- cation of the roles of microtubule motors and their associ- ated proteins in the translocation of Golgi-targeted/derived transport intermediates and in the maintenance of the Golgi's centrosomal location [8°°]. As discussed in this review, such work is revealing the molecular mechanisms underlying the interactions of cytoske!etal motor proteins with organelle membranes. Evidence has been obtained for a spectrin-based matrix for attachment of motor proteins to membranes and for regulation of membrane transport to and from the Golgi complex Ro le o f mic ro tubu les in ER- to -Go lg i t ra f f i c In lower eukaryotic cells that lack the cytoskeletal machin- ery necesssary for spatially segregating Golgi membranes within the cytoplasm (e.g. intracellular parasites), the Golgi complex is often found next to ER exit sites. In such cells the transitional ER and pre-Golgi membranes form the first cisternae of the Golgi stack [9] and transport intermediates move by random diffusion from the ER to the Golgi. In higher eukaryotic cells, however, the ER and Golgi complex are spatially segregated. The ER's network of branching membrane tubules extends outward along microtubules throughout the cell [10], while Golgi cisternae are clustered around microtubules near the MTOC [7]. Transport intermediates that arise from peripheral ER sites, therefore, must often travel considerable distances (up to 201am) to reach the Golgi complex. Work from many labs has revealed that pre-Golgi struc- tures translocate from peripheral to central sites in the cell along microtubule tracks [11,12",13°°]. The most compelling data has come from the work of Presley et al. [12 °°] and Scales et al. [13°°]. These authors visualized ER-to-Golgi trafficking in mammalian tissue culture cells using temperature-sensitive vesicular stomatitis viral G protein (VSVG) tagged with green fluorescent protein (GFP) [14] (GFP). VSVG reversibly misfolds and is retained in the ER at 40°C; however, upon shift to Cytoskeletal proteins and Golgi dynamics Liptoincott-$chwartz 53 320C, it exits the ER and is transported to the Golgi complex and then to the plasma membrane [15]. When VSVG-GFP expressing cells were shifted from 400C to 32°C, VSVG-GFP was found to rapidly enter a variety of differently shaped pre-Golgi structures, which had formed de novo at nonfixed random sites throughout the peripheral ER system [12",13"]. These structures are known from ultrastructural studies not to be small vesicles, but elaborate 0.5!am diameter membrane complexes of tubules and vesicles [16,17]. Having formed, the complexes remained at peripheral sites for only a short period before moving as a single entity in a stop-and-go manner to the cell center. Microtubules were shown to be required for the move- ment of pre-Golgi structures--when microtubules were depolymerized, pre-Golgi structures remained at stationary peripheral ER exit sites [12"',13"']. Transport along mi- crotubules was solely minus-end-directed, with maximum velocities of 1.4~m/s. Pre-Golgi structures were often stretched into elongated forms, suggesting they were pulled through a cytoplasmic meshwork by the action of a microtubule motor complex. Upon reaching the Golgi region, the structures appeared to fuse directly with the Golgi complex. Dynein/dynactin-mediated translocation of pre-Golgi structures Cytoplasmic dyneins are obvious candidates for pow- ering the inward translocation of pre-Golgi transport intermediates along microtubules. These motor proteins are minus-end-directed, microtubule-dependent ATPases, that perform a variety of functions within cells, including that of organelle motility [18]. Cytoplasmic dyneins alone, however, do not effectively translocate organelles, but require an accessory protein complex known as dynactin [19]. The dynactin complex contains an F-actin like filament known as Arpl, which is capped at one end by actin-capping-protein, and at the other by a 62kDa subunit [20]. It also contains the plSO Glued protein, a filamentous projection which protrudes from one end of the Arpl filament [21,22]. In addition, a 50kDa protein known as dynamitin/p50 is present somewhere in the complex [23",24]. Dynamitin/p50 has been shown to regulate both the assembly of the dynactin complex and its interaction with dynein in mitotic cells [23"]. When dynamitin is overexpressed in such cells, the dynactin complex fails to assemble correctly and attachment of cytoplasmic dynein to chromosome-associated kinetochores isreduced [23"]. This is thought to occur because p150 Glued which interacts with cytoplasmic dynein, becomes separated from the Arpl filament, which is responsible for kinetochore attachment. Without dynein as a motor, kinetochores cannot slide along microtubules toward spindle poles. Because dynamitin/p50 overexpression interferes with dynactin/dynein complex assembly and activity without significantly perturbing microtubule organization or stabil- ity (except after long-term expression), it can be used as a useful tool for dissecting the steps in membrane trafficking that are mediated by this motor complex [25"']. To test whether the dynein/dynactin complex is required for the inward translocation of pre-Golgi membranes, Presley et al. [12"'] overexpressed dynamitin/p50 in COS cells. They found that pre-Golgi structures failed to translocate inward along microtubules to the Golgi complex and remained stationary in the cell periphery. This finding strongly suggests that the dynein/dynactin complex powers the inward movement of pre-Golgi membranes. The localization of antibodies to dynein heavy chain 2 (DHC2) to these membranes [26"], further suggests that this isotype of dynein interacts with dynactin in the transport process. It is not known whether dynein and/or microtubules remain associated with stationary pre-Golgi structures during overexpression of dynamitin/p50. This question needs to be addressed in order to understand how the dynactin/dynein motor complex, pre-Golgi structures and microtubules associate with each other. One possible mode of association among these elements is if dynactin anchors dynein to pre-Golgi membranes, by analogy with its role in anchoring dynein to kinetochores in mitotic cells [23"]. Whether this occurs by direct binding of dynactin to membrane receptors on the surface of pre-Golgi structures or indirectly through interactions with peripheral proteins which wrap around pre-Golgi elements is not known. An appealing feature of an indirect interaction is that it could explain how tubulovesicular clusters that comprise pre-Golgi intermediates behave as a single membrane entity during their translocation to the Golgi complex. If a peripheral matrix were to tether the membrane tubules/vesicles together as a single unit, dynactin could interact with this matrix rather than the surface of individual tubules/vesicles. With the help of dynein, it could then move the tubulovesicular mass in a synchronized fashion along microtubules to the Golgi region. Roles of Golgi spectrin and ankyrin On the basis of recent morphological and biochemical work, one set of peripheral proteins that might serve as a meshwork on pre-Golgi and Golgi membranes to anchor dynactin/dynein is spectrin and anykyrin [27"']. Spectrin is an actin-binding protein which in many cell types forms a plasma membrane-associated cytoskeleton [28",29,30]. It generates a planar array or lattice, in conjunction with actin, at the cytoplasmic face of plasma membranes and binds to a subset of integral membrane proteins (e.g. band 3, Na+/K+ ATPase etc.) via ankyrin and other proteins [31]. In this manner, the spectrin meshwork gives structural integrity and mechanical stability to the membrane. In addition, it has been shown to set up microdomains of restricted protein diffusion in the plasma membrane and 54 Cytoskeleton Figure 1 Plasma membrane "1- ER 3olgi-to-ER carrier Golgi - ~C- Plus end i + cartier Current Opinion in Cell Biology Model for the organization of the transport pathways leading into and out of the Golgi complex and the role of microtubules. The Golgi complex is centered around the microtubule-organizing-center (MTOC). Microtubules form a uniformly polarized array, with the MTOC at the hub. These microtubules serve as tracks for membrane traffic. ER-to-Golgi transport of pre-Golgi structures is minus-end-directed, with carriers moving toward the Golgi along microtubules. Golgi-to-ER transport and transport of structures to the plasma membrane (PM) are plus-end-directed, with transport structures moving toward the periphery. in so doing generate specialized regions of the plasma membrane [32]. The presence of a spectrin-based matrix associated with Golgi and pre-Golgi membranes has been demonstrated by Beck etal. [33] and Devarajan and Morrow [34°°]. They observed an isoform of lS-spectrin associated with pre- Golgi/Golgi membranes which had biochemical properties distinct from those of plasma membrane ~-spectrin. In ad- dition, isoforms of the spectrin-associated protein ankyrin were also found localized to these membranes [34"°,35°°]. Biochemical experiments showed that Golgi 13-spectrin and ankyrin associated in higher order oligomeric protein complexes that also include membrane proteins [34°°]. These findings suggest that [B-spectrin and ankyrin form a meshwork on the surface of pre-Golgi/Golgi membranes, which is analogous to that formed by their counterparts on erythocyte plasma membranes. Does this Golgi matrix serve to mediate associations between pre-Golgi/Golgi membranes and motor protein complexes? Although we do not yet know for sure, recent data support this possibility. Holzbaur and colleagues [36 °°] found that Golgi [3-spectrin co-immunoprecipitated with dynactin subunits from rat brain cytosol and co-eluted with dynactin using affinity chromatography [36°°]. More- over, in cells overexpressing Arpl, Golgi I]-spectrin as well as Golgi membranes co-localized with the long filamentous Arpl polymers that formed in the cytoplasm. These data raise the possibility, therefore, that dynactin functions as Cytoskeletal proteins and Golgi dynamics Lippincott-Schwartz 55 Figure 2 Microtubule a spectnn epectrin DH( Ankyrin ARP1 dynactin Veside Ankyrin Current Opinion in Cell Biology Model for interaction of dynactin/dynein with microtubules and membrane intermediates. The dynactin complex has been suggested to power the translocation of pre-Golgi structures along microtubules. The complex comprises dynein, the dynein intermediate chain (DIC), p150 Glued, dynactin (p50/dynamitin and p62) and the F-actin-like filament Arpl. The anchoring of the dynactin complex to membranes may be mediated by an ankyrin/spectrin meshwork. DHC, dynein heavy chain. a bridge to link dynein to organelles via the former's association with spectrin [37]. How might these associations be regulated? Beck et al. [35 "°] found that treatment with brefeldin A (BFA), a drug that prevents membrane export from the ER and disassembles the Golgi complex, results in the dissociation of 13-spectrin and ankyrin from pre-Golgi/Golgi mem- branes. The kinetics for this were similar to dissociation by BFA of other coat proteins that constitutively cycle on and off membranes. These include coatomer (COPI) and ADP ribosylation factor (ARF), which is a small molecular weight GTP-binding protein [38]. This finding has led to the suggestion that the Golgi-associated spectrin mesh- work also undergoes a constitutive cycle of assembly onto and dissociation from Golgi membranes [35°']. Add-back and depletion studies with ARF showed that it could recruit spectrin onto Golgi membranes [39]. Since spectrin has low affinity for negatively charged phospholipids like phosphatidyl inositol 4,5 bisphosphate (PIP z) [30,40] and ARF stimulates an increase in local Golgi membrane PIP e concentrations [40], spectrin binding to pre-Golgi/Golgi membranes could be regulated by ARF activity. Golgi-to-ER trafficking: roles of microtubules and kinesin A pathway carrying Golgi membrane back to the ER is known to exist [41]. This pathway recycles membrane machinery necessary for forward secretorytraffic and maintains the surface area of the ER in the face of membrane outflow into the secretory pathway. Several proteins have been shown to follow the Golgi-to-ER pathway. These include ERGIC53 (a lectin-binding pro- tein) [42] and the KDEL receptor [43]. Recently, Cole et al. [44] showed that entry into this pathway may be a constitutive property of proteins residing in the Golgi 56 Cytoskeleton complex. In this study Golgi-resident chimeras containing the temperature-sensitive lumenal domain of VSVG (in- cluding VSVG-KDEL-receptor and VSVG-TGN38) were found to redistribute back to the ER upon temperature shift to 40°C. VSVG itself did not recycle back to the ER under these conditions, but was transported to the cell surface. Thus, only chimeras that constitutively cycle between the ER and the Golgi complex were misfolded and became trapped in the ER upon shift to 40°C. The nature of Golgi-to-ER transport intermediates and the role of microtubules and motor proteins in facilitating their transport through the cytoplasm has only begun to be addressed. Recent work using GFP-tagged KDEL receptors localized to Golgi membranes suggests that thin membrane tubules, rather than vesicles, serve as transport intermediates for Golgi-to-ER transport [45]. These inter- mediates in no way resembled with the large, pleiomorphic pre-Golgi structures involved in ER-to-Golgi traffic. In addition, they moved in the opposite direction along microtubules. In time-lapse imaging experiments, Golgi tubules containing the GFP-tagged KDEL receptor were observed extending toward the plus ends of microtubules [45]. Upon detaching from Golgi elements, the tubules translocated along microtubule tracks, at rates of 0.6 ~tm/s, to the cell periphery--where they later disappeared from. view, perhaps as a result of fusion with the ER. In cells treated with BFA, which enhances Golgi membrane recycling to the ER [45], Golgi membrane tubules with a similar appearance formed, but more rapidly. These tubules failed to detach from Golgi structures and formed an elaborate network of membranes extending throughout the cytoplasm. Fusion of one or more of the Golgi tubules with ER membranes after 5-10 min of BFA-treatment led to the rapid redistribution of Golgi membrane and content into the ER [45]. Microinjection of antibodies to the kinesin heavy chain blocks the extension of Golgi membrane tubules along microtubules in BFA-treated cells [46]. This suggests that kinesin is required for the plus-end-directed movement of these membranes along microtubules. Since antibodies to kinesin label predominantly pre-Golgi and Golgi membranes [46--48] (which both actively cluster towards the minus ends of microtubules), kinesin could travel with these membranes in an inactive state before becoming activated for microtubule plus-end-directed retrieval of membrane to the ER. How!kinesin motor activity is selectively turned on and off as it cycles with membranes between the ER and Golgi complex is not known. Kinesin, unlike dynein, does not appear to require an extensive set of accessory molecules in order to translocate organelles [49]; once attached to membranes, possibly via the transmembrane protein kinectin [50,51], kinesin appears to be capable of translocation along microtubules in the presence of ATP. Changes in the phosphorylation status of kinesin [52,53] and/or the presence of molecules that interfere with its attachment to membranes or microtubules [49], however, could modulate its ability to translocate cargo. Given that BFA enhances kinesin-based plus end-directed activity of Golgi membranes [45] and inhibits dynein-based minus-end-directed translocation of pre-Golgi membranes [13"], a reciprocal relationship between dynein and kinesin activity on these membranes is possible. The spectrin/ankyrin Golgi cytoskeleton or COPI proteins, both of which dissociate from pre-Golgi and Golgi membranes in BFA-treated cells [27",38], could directly or indirectly affect the activities of these motor proteins. Golgi membrane remodeling in response to microtubule depolymerization The normal distribution of Golgi membranes within cells represents a dynamic steady state of forward and recycling pathways connecting the Golgi and ER [8"']. Thus, any perturbation of forward traffic into the Golgi complex without a corresponding effect on recycling results in the redistribution of Golgi material to the site of perturbation. An excellent example of this is fragmentation of the Golgi complex observed in nocodazole-treated cells [8"',54], cells microinjected with antibodies to DHC2 [26"*], and cells in which dynamitin/p50 has been overexpressed [25**]. All of these treatments block the inward trans- location of pre-Golgi intermediates along microtubules without having significant effects on Golgi-to-ER traffic. Since Golgi enzymes constitutively cycle between the ER and the Golgi complex via these forward and reverse pathways [44], they accumulate at ER exit sites when forward trafficking along microtubules is inhibited [8"']. Golgi enzymes and other components that accumulate at these sites over time rebuild Golgi s tacks- -a process that results in the formation of hundreds of Golgi fragments which are distributed throughout the cell [8"']. Prior to Golgi fragmentation, secretion is significantly inhibited within cells because pre-Golgi structures car- rying secretory cargo derived from the ER cannot reach the centrosomally-localized Golgi complex. After Golgi fragmentation, secretion occurs at a normal rate, but delivery to the cell surface becomes randomized. Traffic from the Golgi to the plasma membrane: roles of microtubules and actin-based motors Proteins and lipids leave the Golgi complex at the trans Golgi network (TGN) where they are sorted into pathways to lysosomes, secretory granules and the plasma membrane. The role of cytoskeletal elements in these processes have only begun to be worked out. Kaether et al. [55] and Wacker et al. [56] have used GFP-tagged secretory protein chromogranin B (hCgB-GFP) to follow constitutive secretory traffic from the Golgi to the plasma membrane in Vero cells. Post-Golgi intermediates containing hCgB-GFP were observed moving away from Cytoskeletal proteins and Golgi dynamics Lippincott-Schwartz 57 the Golgi region along microtubule tracks at velocities of 1 I, tm/sec. In contrast to pre-Golgi structures, the post- Golgi structures moved bidirectionally along microtubules with many structures reversing directions within short time periods (<45s). Microtubule disruption slowed the delivery of hCgB-GFP to the plasma membrane, but did not abolish it. This suggests microtubules facilitate, but are not required for, transport from the Golgi to the plasma membrane. Recent studies have implicated the actin-based motor myosin II in the membrane dynamics occurring at the TGN. Mtisch eta/. [57 °-] showed that the TGN-associated protein p200 [58,59••], is myosin lI, and was involved in the release of transport intermediates containing the viral glycoprotein VSVG from the TGN. The membrane binding properties of p200/myosin are similar to those of coat proteins involved in vesicle assembly [57••,58]. Binding was dependent on GTP, or its nonhydrolyzable analog GTPTS, and was stimulated by aluminium fluoride. Moreover, BFA prevented myosin II recruitment to the Golgi complex [57°°]. These results suggest that myosin II is either part of a TGN coat complex or is recruited to the site of membrane budding, after coat assembly, to help detach transport intermediates. Miisch et al. [57 °-] found that formation of transport intermediates containing VSVG in vitro was inhibited by agents which prevent interactionof myosin with actin. This suggested that it was the actin-dependent ATPase activity of myosin II that was involved in membrane budding. Myosin II's role at the TGN, therefore, might be to regulate actin filaments involved in pulling transport intermediates off the TGN. Alternatively, it could promote actin rearrangement to allow coat protein recruitment for membrane budding. The question of whether myosin lI also regulates the dynamics of the spectrin-based Golgi skeleton to directly or indirectly faciliate membrane trafficking remains to be addressed. Conclusions The Golgi complex is a steady-state membrane system which depends on the balance of membrane inflow and outflow pathways. In higher eukaryotic cells, these pathways are microtubule-facilitated and underlie the subcellular distribution and size of the Golgi complex. Recent studies have focused on clarifying the roles of microtubule motor proteins, such as cytoplasmic dynein, kinesin and myosin, in mediating the translocation of Golgi transport intermediates. New results suggest that a Golgi-associated spectrin cytoskeleton may have an important role in providing a structural platform for the attachment of these molecules. Understanding how the microtubule-based and spectrin-based cytoskeletal systems associated with the Golgi complex interact and serve to regulate membrane traffic into and out of this organelle are questions ripe for future work. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest • • of outstanding interest 1. Mellman I, Simons K: The Golgi complex: in vitro veritas? Cell 1992, 68:829-840. 2. Lippincott-Schwartz J: Bidirectional membrane traffic between the endoplasmic reticulum, Golg apparatus. Trends Cell B/o/ 1993, 3:81-88. 3. Cole NB, Lippincott-Schwartz J: Organization of organelles and membrane traffic by microtubules. Curt Opin Cell B/o/1995, 7:55-64. 4. Luby-Phelps K, Castle PE, Taylor DL, Lanni F: Hundered diffusion of inert tracer particles in the cytoplasm of mouse 3T3 cells. Proc Nat/Acad Sc/USA 1997, 84:4910-4913. 5. Rogalski hA, Bergmann JE, Singer S J: Effect of microtubule assembly starus on the intracellular processing and surface expression of an integral membrane protein. J Cell Biol 1984, 99:1092-1100. 6. Thyberg .I, Moskalewski S: Microtubules and the organization of the Golgi complex. Exp Cell Res 1985, 159:1-6. 7. Ho we, Allan VJ, van Meer G, Berger EB, Kreis TE: Reclustering of scattered Golgi elements occurs along microtubules. Eur J Cell Bio11989, 48:250-263. 8. Cole NB, Sciaky N, Marotta A, Song J, Lippincott-Schwartz J: *, Golgi dispersal during microtubule disruption: regeneration of Golgi stacks at peripheral endoplasmic reticulum exit sites. Mol Bio/ Cell 1996, 7:631-650. This study analyzes the process whereby Golgi membranes 'fragment' during microtubule depoiymerization by nocodazole. Using a variety of techniques the authors show that Golgi fragmentation arises from a slow but consti- tutive flux of Golgi resident proteins through the bidirectional membrane pathways connecting the ER and Golgi complex. Since transport of recycling intermediates to the ER is unaffected by microtubule disruption, whereas translocation of pre-Golgi structures into the centrosomal region is blocked, when microtubules are disrupted, Golgi enzymes accumulate at ER exit sites over time. 9. Sheffield HG, Melton M J: The fine structure and reproduction of Toxoplasma gondii. J Paras/to/1968, 54:209-226. 10. Terasaki M, Chen LB, Fuguwara K: Microtubules and the endoplasmic reticulum are highly interdependent structures. J Cell B/o/ 1986, 103:1557-1568. 11. Saraste J, Kuismanen E: Pathways of protein sorting and membrane traffic between the rough endoplasmic reticulum and the Golgi complex. Semin Cell B/o/1992, 3:343-355. 12. Presley JF, Cole NB, Schroer TA, Lippincott-Schwartz J: ER-to- ** Golgi transport visualized in living cells. Nature 1997, 389:81- 85. The viral glycoprotein ts045 VSVG tagged with green fluorescent protein (VSVG-GFP) was used to study ER-to-Golgi traffic in living cells. When visualized at the permissive temperatures, upon export from the ER, VSVG- GFP became concentrated in many differently shaped pre-Golgi structures, which then translocated inwards, along mictoubules, toward the Golgi com- plex. The motor complex of dynein/dynactin was required for this movement. Because pre-Golgi structures are large tubulovesicular masses rather than individual vesicles, these results focus attention on the role of such structures in Golgi biogenesis and trafficking. The study reported the first visualization of ER to Golgi traffic in real time. 13. Scales S J, Pepperkok R, Kreis TE: Visualization of ER-to-Golgi ** transport in living cells reveals a sequential mode of action for COPII and COPI. Cell 1997, 90:1137-1148. This study characterized ER-to-Golgi traffic, again using VSVG-GFP, and addressed the potential roles of COP1 and COPII. Both of these periph- eral proteins were associated with pre-Golgi structures at the time of their formation. 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