The Golgi Complex: In Vitro Veritas? Review
Abstract
Understanding thestructure and function of theGolgi comiplex has proved to be among the more challenging probllems in cell biology. The last several years have turned out Ito be particularly exciting in this respect since they have Iyielded new insights and ideas at an increasingly rapid ipace. This period of advance has largely been due to the Idevelopment of powerful new biochemical, morphological, #and genetic approaches to unraveling the complexities of 'this organelle. While much remains to be discovered, the Iproblem now is how to integrate this wealth of information.
'To see if this is possible, we will first summarize how the lslolgi is commonly believed to work and then evaluate the lstrength of the evidence that underlies these views.
iplex has proved to be among the more challenging probllems in cell biology. The last several years have turned out Ito be particularly exciting in this respect since they have Iyielded new insights and ideas at an increasingly rapid ipace. This period of advance has largely been due to the Idevelopment of powerful new biochemical, morphological, #and genetic approaches to unraveling the complexities of 'this organelle. While much remains to be discovered, the Iproblem now is how to integrate this wealth of information.
'To see if this is possible, we will first summarize how the lslolgi is commonly believed to work and then evaluate the lstrength of the evidence that underlies these views.
Present View of the Golgi 'The Golgi complex is essentially a carbohydrate factory. In Transfer of the VSV G protein from Golgi membranes in the infected to the uninfected cells was monitored by a glycosylation event that could be carried out by only the uninfected recipient, which was chosen to complement the glycosylation defect of the infected cell. These observations indicated that efficient transfer of VSV G protein occurs between spatially distinct Golgi stacks. This is most easily explained byamechanism involving VSVG proteincontaining transport vesicles that form from the infected cell Golgi, diffuse through the heterokaryon's cytosol, and fuse with Golgi membranes in the uninfected cell. Conceivably, the same transport vesicles could mediate intercisternal transport within a single Golgi stack.
The major limitation of these data is that they are indirect. They do not identify the vesicles nor do they definitely establish their site of origin. One cannot exclude the possibility that the G protein moved by lateral diffusion through tubules connecting the heterologous Golgi stacks, which otherwise remain separated during the assay. Furthermore, even the existence of vesicles involved in inter-Golgi transport does not demonstrate that they have a similar role in intercisternal transport within a single stack, as is assumed by our current view of the Golgi.
In Vitro Transport through the Golgi and the Role of Carrier Vesicles The most extensive series of considerations favoring the role of transport vesicles in intra-Golgi transport comes from experiments designed to reconstitute transport in vitro. That such transport can be achieved has been demonstrated over the past several years by the pioneering work of Rothman and coworkers. In their approach, Golgi fractions prepared from VSV-infected GlcNAc transferase-deficient 158 cells ("donor Golgi") are incubated with similar fractions from uninfected wild-type cells ('acceptor Golgi"). Upon the addition of ATP and cytosolderived factors, there is a progressive appearance of [3H]GlcNAc-labeled (Balch et al., 1984) and endo H-resistant (Fries and Rothman, 1981) VSV G protein, reflecting its transfer from mutant "donor" to wild-type "acceptor" membranes.
This transfer has also been dissected kinetically into two major stages: an early stage presumed to correlate with the budding of transport vesicles from the donor Golgi, and a late stage reflecting the docking and subsequent fusion of the vesicles with the acceptor Golgi. The early and late stages also differ in their requirements and inhibitor sensitivities (Balch et al., 1984; Rothman and Orci, 1990) . Given that VSV G protein in early Golgi compartments was transported with the highest efficiency (Fries and Rothman, 1981) the transport event assayed was postulated to reflect the formation of vesicles from cis (early) cisternae and the fusion of these vesicles with medial cisternae (which contain the GlcNAc transferase) (Rothman and Orci, 1990) (Figure 4) . However, the identi-DONOR ACCEPTOR Fllgure 4. Two Models to Account for Donor-Acceptor Connections in Inter-Golgi Transport In Vitro (A) Vesicular transport, as predicted by the current view of the Golgi alid by the work of Rothman and colleagues (Rothman and Orci. 1990) . (ES) Vesicle-independent direct fusions between donor and acceptor compartments (CGN-CGN, medial-medial, and TGN-TGN), perhaps mediated by tubular extensions. While these tubules may be amplified by BFA. as described in the text, they may exist and function even in the absence of the drug. ties of the donor and acceptor cisternae have not yet been established, nor can kinetic analysis alone provide a unique interpretation, especially when transport is monitored indirectly by linkage to glycosylation, which itself is a kinetically complex series of reactions (see above).
Analysis of this Golgi transport system has already led to the discovery of several discrete protein factors whose a.ctivities are required to mediate the assay (Rothman and Orci, 1990) . Some of these represent the mammalian homologs of yeast genes known to be involved in intracellular transport in intact cells (Wilson et al., 1989; Clary et al., 1990) . One important factor, designated NSF (NEM-.sensitive fusion protein), is homologous to the yeast sec78 gene product and is thought to act at a late stage in transport, at the time when transport vesicles fuse with the acceptor membranes Orci et al., 1989; Rexach and Schekman, 1991) both in ER to Golgi and in Golgi as well as in post-Golgi transport (Graham and Emr, 1991) . The precise function of NSF, however, remains unknown. Another factor, designated SNAP (for soluble NSF attachment protein), is homologous to the yeast sec77 gene product and is thought to be involved in NSF binding to membranes (Clary et al., 1990) .
The strongest evidence for the existence of a vesicular intermediate in the in vitro transport reaction has been provided by the identification and isolation of the presumed carrier vesicles themselves. During the early stagesof the reaction, isolated Golgi membranes generate an array of coated tubules, buds, and vesicles (Orci et al., '1986 ). The vesicles have been isolated (after elution from l:heir membrane-bound state by high salt), and their coats have been found to contain several unique components . One of these, B-COP, was previously identified by Kreis and coworkers as a 110 kd peripheral protein tightly associated with the Golgi (Allan and IKreis, 1986; Duden et al., 1991b; Serafini et al., 1991) . These non-clathrin-coated vesicles are good candidates ,for carrier vesicles. First, they contain VSV G protein, the iprotein being transported (Orci et al., 1986 Serafini let al., 1991) . Next, conditions that block the transport reac-tion in general result in the accumulation or lack of formation of vesicles. Most interestingly, treatment of cells or isolated membranes with the nonhydrolyzable GTP analog, GTPyS, both blocks glycosylation of the VSV G protein and results in the accumulation of VSV G protein-containing coated buds and vesicles . Similar results were obtained using AIF4, an inhibitor of conventional GTP-binding "G proteins" Kahn, 1991) . The stage at which these inhibitors act remains unknown (Donaldson et al., 1991 b) .
In spite of this large amount of correlative evidence, additional data will be required before it is certain that the coated vesicles serve as unique and obligatory transport intermediates either in vitro or in intact cells. Perhaps the most important piece of missing information pertains to whether the vesicles have a composition consistent with their presumed transport function. For example, it will be important to confirm that the Golgi vesicles do not contain resident proteins, e.g., glycosyltransferases, that are not intended for transport. The analogous point is well established for plasma membrane clathrin-coated vesicles. Many examples are known where proteins destined for coated vesicle-mediated endocytosis accumulate at coated pits, while those that are not efficiently internalized do not enter coated pits (Pearse and Robinson, 1990) .
Another critical unknown is a demonstration that the Golgi coated vesicles are functional in vitro, i.e., that VSV G protein-containing vesicle fractions can be used to reconstitute transport when added to acceptor Golgi. The analogous experiment has recently been accomplished for a less well-characterized vesicle fraction that mediates transport from the ER to the Golgi in yeast (Groesch et al., 1990; Rexach and Schekman, 1991) . Thus, in principle, this approach should be possible. However, with all such experiments, it will be important to ensure that the coated vesicle fractions used are not contaminated with uncoated, fusion-competent membranes.
BFA Blocks Vesicle Formation without Blocking Transport A reason for examining the features of the Golgi vesicles so carefully is indicated by a recent paper that raises the possibility that, at least under certain conditions, these vesicles may not be required for transport in vitro . In this work, the effect of BFA on the Golgi transport assay was determined. BFA is a macrocyclic fungal antibiotic that blocks the transport of membrane and secretory proteins through the Golgi (Takatsuki and Tamura, 1985; Misumi et al., 1986) . In intact cells, the drug causes a dramatic and rapid retrograde transport of Golgi components back to the ER via a system of tubular extensions (Lippincott- Schwartz et al., 1990) . The mechanism of BFA action is unknown, but one intriguing property is its ability to rapidly, and reversibly, trigger the dissociation of B-COP (Donaldson et al., 1991a; Klausner et al., 1992) .
When isolated Golgi was treated with BFA, P-COP was no longer associated with the membranes nor were any coated buds or vesicles detected . Nevertheless, the transfer of VSV G protein from donor to acceptor Golgi continued with similar kinetics and efficiency as in untreated controls. The only difference was that transfer in BFA-treated Golgi lost its sensitivity to GTPyS inhibition.
There are two possible interpretations of these results. The first is that coated vesicles that form in the assay system are not obligatory intermediates in the transport event, even in the absence of the drug. Alternatively, BFA may induce an aberrant form of transport that nevertheless utilizes enzymatic machinery required for vesicular transport in the absence of the drug. It is at present impossible to distinguish between these two possibilities. However, BFA-treated Golgi in vitro appear to form tubular extensions that may serve to interconnect donor and acceptor Golgi stacks, thus resulting in transfer to the acceptor across these tubular bridges . This possibility is appealing since it could reflect the BFA-induced tubulation of the Golgi seen in intact cells. However, additional electron microscope immunocytochemistry will be required to establish continuous interconnections between antigenically distinct Golgi elements.
Vesicular Transport or Golgi Fusion? The fact that "transport" (i.e., G protein glycosylation) can occur in the absence of vesicle formation raises a number of important questions: What transport step(s) is actually being measured in vitro? What is the relationship of the events reconstituted in vitro to those found in intact cells? Is transport in vitro ever completely dependent on vesicle formation, even in the absence of BFA? If vesicles are not involved, is transfer from donor to acceptor Golgi mediated by direct fusion, perhaps via tubular extensions? As mentioned above, it is unclear whether the G protein is transferred across a predicted compartment boundary (donor CGN to acceptor medial) or within a single compartment (donor medial to acceptor medial).
It is becoming increasingly clear that all membranebound organelles have a propensity to form tubules in intact cells and in vitro, with or without BFA treatment. For example, the ER (Lee and Chen, 1988), mitochondria (Johnson et al., 1980) peroxisomes (Yamamoto and Fahimi, 1987) endosomes (Hopkins et al., 1990; Tooze and Hollinshead, 1991; Hunziker et al., 1991b; Lippincott-Schwartz et al., 1991) , and lysosomes (Swanson et al., 1987; Lippincott-Schwartz et al., 1991) are all capable of forming dynamic tubular membrane networks. One wellstudied example is the early endosome, the structure of which varies in different cell types (Marsh et al., 1986; Tooze et al., 1991) . Cell-free assays have documented that the early endosome elements can fuse avidly with each other (Gruenberg and Howell, 1989) . Other examples of processes involving homotypic fusion in vitro are the assembly of the nuclear envelope (Burke and Gerace, 1986) and the formation of ER (Dabora and Sheetz, 1988) .
The Golgi complex is also a dynamic structure that can be disassembled by microtubule depolymerization into fragments that remain functional despite being dispersed throughout the cytoplasm (Thyberg and Moskalewski, 1985) . Upon microtubule reassembly, the Golgi fragments rapidly reassemble and assume their characteristic cisternal organization (Ho et al., 1989) . A similar series of events must occur during mitosis when the Golgi fragments into populations of small vesicles and tubules (Lucocq et al., 1989) . During telophase, these fragments reassemble.
Given that homotypic fusion occurs so commonly, it would be premature to dismiss the possibility that the intra-Golgi transport activity observed in vitro actually reflects a process of regulated tubule formation and/or direct fusion of donor and acceptor Golgi elements (Figure 4) . Indeed, even in untreated Golgi, both in vitro and in intact cells, tubules can be found that are similar to those observed in BFA-treated Golgi (Hermo et al., 1980; Braell et al., 1984 [see Figure 8b in this reference]; Griffiths et al., 1985) . That a direct fusion event is possible is also suggested by the fact that the predicted diffusion coefficient of intact Golgi stacks is ~1 O-fold less than that of the much smaller coated vesicles (N. Ktistakis, unpublished data). Thus, one might expect a decrease in the kinetics of VSV G protein transfer if BFA were to switch the signal generated in vitro from a vesicle-dependent phenomenon (as is presumed to occur in the absence of the drug) to a direct, tubule-mediated fusion event. However, G protein is transported from donor to acceptor with the same kinetics in the presence or absence of BFA . Finally, the fact that the two N-linked chains of VSV G protein are processed simultaneously in vitro as opposed to sequentially in permeabilized cells may also indicate that compartmental boundaries may not be strictly preserved, i.e., that direct fusion may occur, during the cell-free assay (Schwaninger et al., 1991) .
The minimal model that we discussed (see Figure 3 ) predicts only two functional and presumably physical discontinuities in theGolgi: from theCGN to the medial Golgi, and from the medial Golgi to the TGN. Thus, according to this view, transport through the Golgi involves two intercompartmental transfers, reflecting transfer from the site of entry to the site of glycosylation, and from the site of glycosylation to the site of exit.
Considering available data, we consider it likely that the intercompartmental transfers between CGNlmedial and medial/TGN boundaries occur in vivo via vesicular carriers While the putative transporters remain to be identified, the P-COP-containing vesicles Serafini et al., 1991) are obvious but unconfirmed candidates, whether or not they are ultimately found to function as carrier vesicles in in vitro Golgi assays. The tubular interconnections known to occur in the Golgi may also play a role in intercompartmental transfer, but we view these as probably being more important for establishing and maintaining intracompartmental links that serve to interconnect functionally identical Golgi compartments and to mix their contents. These tubular connections may break and fuse continuously, accounting for the dynamic behavior of the Golgi complex after microtubule disassembly and reassembly. However, if their formation is tightly controlled by a regulated process of assembly and disassembly of the f&COP-containing Golgi coat, even tubule-based intercompartmental transfer is not inconceivable. Moreover, whether cell-free assays of Golgi function reflect such an intercompartmental transfer, or intracompartmental transfer between wild-type and mutant Golgi, remains to be solved. Only further characterization of the machinery responsible for intra-Golgi transport will decide between these two possibilities. Nevertheless, based on the evidence both from the in vitro assays and from yeast genetics, it is clear that the few proteins identified thus far, such as NSF and SNAP, are generally important elements, irrespective of the precise pathways or whether transport occurs via vesicles or tubules.
Is Transport through the Golgi Selective or Nonselective? Having considered the evidence for the compartmental organization of the Golgi and for the role of carrier vesicles in transport, we come to the last of the three general elements underlying our view of Golgi function, namely, that transport of passenger proteins through the Golgi is inherently nonselective (Pfeffer and Rothman, 1987) . The evidence for this view is largely indirect or negative, in that efforts to identify discrete signals on proteins that are required for forward transport have thus far been unsuccessful. On the other hand, as discussed above, there is now considerable evidence in favor of several distinct "retenlion signals" that effectively prevent forward transport of membrane or lumenal proteins after reaching their prescribed destinations in the ER or various Golgi compartments (Pelham, 1991) . If this view is correct, selectivity in I.ransport through the secretory pathway may occur by "default," i.e., transport of passenger proteins proceeds owing to the absence of a retention signal. This concept, however, is not necessarily incompatible with the existence of selective signals for forward transport. It is clear that such signals exist and play an important role in directing the traffic of proteins as they leave the TGN. Examples include the mannose g-phosphate residues that specify transport of hydrolytic enzymes to lysosomes and Mellman, 1989) and the cytoplasmic domain determiinants that target newly synthesized membrane proteins to ithe basolateral surface of polarized cells (Hunziker et al., 1991a; Brewer and Roth, 1991) . Since the transport of ,fluorescent lipids from the Golgi to the plasma membrane Iin nonpolarized cells occurs very rapidly, it is thought that "constitutive" transport from the Golgi to the cell surface may not require specific signals . ,Analogous results were obtained in experiments in which a tripeptide containing a cognate site for N-linked glycosylation presumably also involved transport from the ER (Wieland et al., 1987; Helms et al., 1990) . While these experiments suggest that signals are not necessary for transport, the fact that they are released with rapid kinetics does not alone establish the absence of such signals.
Experiments showing that different secretory and membrane proteins are transported with different kinetics may indicate that signals or receptors are involved in forward transport (Lodish et al., 1983; Lodish, 1988) . Similarly, the suggestion that newly synthesized viral spike glycoproteins are present in Golgi membranes at a density severalfold greater than in the ER is also consistent with the existence of signal-driven forward transport (Griffiths et al., 1984) . However, both of these observations might also be reconciled with a nonselective mechanism of transport.
Given recent evidence that exit from the ER is linked to the folding of newly synthesized proteins, differential folding rates among proteins may indirectly affect their transport kinetics. Moreover, if the intrinsic rate of transport of glycoproteins through the Golgi is slow relative to the rate of ER exit, then one might also expect a higher concentration of certain passenger proteins in the Golgi.
At present, most of the attention paid to the question of signals in transport concerns ER to Golgi or postQolgi transport. Whether transport through the Golgi complex itself is selective or nonselective is still an open question.
After years of descriptive work, the Golgi complex is slowly starting to reveal its secrets. We have now entered an exciting period of research, during which it will become possible to define the molecular mechanisms responsible for generating and maintaining Golgi structure and function. The first phase is already well under way and has been characterized by a search for essential bits and pieces of the Golgi machinery, a number of which have already been found (NSFlsecl8, aSNAPlsecl7, (J-COP, ARF[Stearnsetal., 19901, rab6p[Goudetal., 1990] ,sec7p [Achstetter et al., 19881, and secl4p [Bankaitis et al., 199Oj) . As we have seen, however, it is at present difficult to know precisely what steps are controlled by each of these components. Nevertheless, the observed conservation of Golgi proteins between S. cerevisiae and mammals is most encouraging for our ability to confirm in living cells the function of components identified in vitro. The combination of cell-free analysis and genetics has proven its worth. The next phase will have to deal with the questions that have arisen. How many Golgi compartments are there? Are compartment boundaries defined by specific protein frameworks? If so, how do they function and how are they regulated? Does transport between Golgi compartments require vesicular carriers? What is the role of tubules? How does the machinery responsible for forward traffic relate to the machinery controlling homotypic fusion? How is specificity of forward and backward traffic regulated? How does lipid composition and organization affect transport? What function does the stack structure have? How do microtubules interact with the Golgi elements? The challenge will be to integrate the information we are now collecting in the context of how the Golgi complex works as a whole.
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