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A synthetic model of intra-Golgi traffic

^a Laboratory of Molecular Neurobiology, Mario Negri Sud Institute, Chieti, Italy

	ABSTRACT

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We present a hypothesis on the mechanisms used by cells to transport cargo through the secretory system. We propose that at least three basic processes coordinately participate in membrane traffic: cisternal maturation–progression, controlled cargo diffusion along transient membrane continuities between different compartments, and a mostly retrograde vesicle-mediated transport. A ‘synthetic’ model based on the combination of these mechanisms can explain both the progression of supramolecular aggregates through the secretory pathway and the fast intra-Golgi transport of conventional cargoes. Analysis of the existing literature shows that the available data are consistent with the proposed model.—Mironov, A., Jr., Luini, A., Mironov, A. A synthetic model of intra-Golgi traffic. FASEB J. 12, 249–252 (1998)

Key Words: intra-Golgi transport • cistern maturation • lateral diffusion • Golgi enzyme recycling

	INTRODUCTION

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ACCORDING TO the current consensus, intracellular transport of proteins and membranes is accomplished by transport vesicles budding from donor and fusing with acceptor compartments. At the same time, the notion is gradually emerging that many pieces of evidence accumulated over the years are difficult to reconcile with an exclusively vesicular paradigm of membrane traffic (1): 1) the observation that supramolecular aggregates (SA) incompatible in size with vesicular carriers are transported from the endoplasmic reticulum (ER) to the Golgi apparatus (2) and through the Golgi (1, 3); 2) the exclusion of at least some soluble cargoes from transport vesicles (4, 5); 3) the lack of direct proof for participation of vesicles in intra-Golgi traffic in cell-free assays (6); and 4) the existence of direct membrane continuities between different compartments of the ER–Golgi system (7–9).

As a consequence, other transport models—diffusion along membranous tubular or saccular connections between compartments and transport by cistern maturation—are beginning to be discussed seriously in the literature after a long period of neglect (1, 3). Each model alone, however, is insufficient to explain all the complexities of intracellular traffic. The goal of this paper is to discuss whether a model based on the combination of mechanisms mentioned above may help to resolve the existing difficulties. Since others have reviewed the vesicular shuttle model (10), we will focus on lateral diffusion and maturation processes.

	LATERAL DIFFUSION ALONG CONTINUITIES BETWEEN HETEROTYPIC COMPARTMENTS

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Continuities have often been observed between Golgi apparatus and the ER as well as between individual Golgi stacks, most of which are linked to one another by intricate networks of tubules and saccules. Moreover, within this complex connecting structure, bridges have been reported to occur not only between homotypic but also heterotypic Golgi cisterns of adjacent stacks (7, 9). Thus, there is a physical basis for cargo molecules to progress toward the plasma membrane by controlled diffusion, as shown in the model in Fig. 1. In this scheme, the basic Golgi transport unit consists of a linear series of cisterns located in different stacks and joined by tubules or saccules in a cis-trans sequence; the whole Golgi structure looks like a large spiral containing several linear transport units. One seeming difficulty of this model is that the lipid and protein gradients known to exist between the Golgi poles could be expected to dissipate through the continuities. However, membrane differentiation in a continuous system is found in other cellular compartments: a well-known example is the presence of different domains (rough and smooth) coexisting in the continuous ER network. Several mechanisms can be envisaged to play a role in maintaining such domains. One could be retrieval of resident proteins, or retention of proteins based on regulated association with the cytoskeleton or other protein scaffolds. Another important mechanism might be control of lateral diffusion by regulated fission and fusion events that would transiently interrupt the membranous connections to allow the system to restore the gradients. Finally, the addition of molecules at one end of the membrane continuum and the removal from the opposite end (1) could contribute to generating the gradients and provide the energy for directional flow of cargo.

View larger version (32K): [in this window] [in a new window]   Figure 1. A synthetic model of intra-Golgi transport. Panels A and B represent two successive stages of the process. A) After arrival from the ER via transport intermediates (TI), soluble cargo (closed circles) quickly diffuses along the membrane continuum (the direction is indicated by small arrows) toward the trans-Golgi network (TGN). Transient interruptions of the continuum that might regulate the diffusion process are not represented for the sake of simplicity. B) The delivery of membranes causes growth of the cis cisterna; concomitantly, this cisterna undergoes maturation into a medial one by acquiring medial Golgi enzymes via retrograde vesicles (RV) from the former medial cisterna (direction indicated by arrowheads), which in turn receives trans enzymes and thus becomes a trans cisterna. In the process, supramolecular aggregates (SA) acquire a more distal position in the stack (direction indicated by large open arrows). Panels C and D represent perpendicular sections of the stack corresponding to stages A and B, respectively. Transport of diffusable cargo occurs tangentially (direction indicated by small arrows in panels A and B) and transport of SA perpendicularly (direction indicated by open arrows in panels A and B and small arrows in panels C and D) to the axis of the stack.

Some aspects of transport by lateral diffusion through continuities are within experimental grasp. For instance, cargo proteins should be demonstrable in membranous connections between heterologous cisterns; even isolation of the connecting intermediates by fractionation techniques should, in principle, be possible. A more specific prediction is that in cells where, due to microtubule disruption, the Golgi complex is fragmented into small separated stack units yet functions normally, connections between heterologous cisterns should still be present though rearranged, presumably in a different fashion. Indeed, a preliminary communication that this might be the case has been reported (ref 1 and references therein). Finally, agents that specifically disrupt intercompartmental connections should inhibit traffic. Although such molecules unfortunately are not yet available, elucidation of the molecular mechanism underlying the formation and maintenance of the membranous bridges will provide the basis for their development. There is, however, a set of data concerning the transport of supramolecular complexes that the lateral diffusion model cannot explain. These observations indicate the existence of another mechanism: cisternal maturation.

	CISTERNAL MATURATION

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Intra-Golgi, and in some cases ER-to-Golgi (2), transport of scales in algae, collagen polymers in many collagen-secreting cells, apo-A-containing chilomicrons and lipoprotein aggregates in hepatocytes, secretory granules in seminal glands, casein submicelles in lactating mammary gland cells, and some types of viruses (1, 3) is not easy to explain by vesicular traffic because these large cargoes are incompatible in size with vesicular transport intermediates. For similar reasons, they are also difficult to reconcile with the lateral diffusion model alone. Only a mechanism involving the progression of a cistern as a whole (where a cis cistern would mature into a medial, and then a trans, cistern) can satisfactorily account for these observations. Since the mechanism of cisternal maturation requires the gradual transfer of resident Golgi enzymes and proteins across successive cisterns in the trans-to-cis direction, a key question is how such transfer is accomplished.

When the cisternal progression mechanism was rejected long ago, researchers thought that resident Golgi enzymes do not move through the Golgi complex. Now there is evidence that Golgi enzymes can move in a retrograde fashion in vivo and in vitro (see refs 1, 3); how they move and, more specifically, in what carrier (or carriers), however, remain unclear. Although retrograde coat protein (COP) -coated vesicles are an obvious candidate and Golgi enzymes (in fact, Golgi enzyme-containing chimerical constructs) can be detected in Golgi vesicles (11), the amounts of these enzymes found in conventionally isolated coated vesicles under normal conditions are low (5, 12). On the other hand, this might not be a problem. If we assume that vesicle fusion and fission are coordinated, and that a vesicle pinching off the cistern (medial, for example) contains a low concentration of enzyme (for instance, 10% of the cisternal levels) whereas another vesicle fusing with the same cistern does not contain medial enzymes (but contains trans enzyme instead), then the concentration of medial enzyme in the cisterns will have decreased after fission of the first vesicle and simultaneous fusion of the second vesicle. Thus, each round of coordinated fission/fusion events of retrograde vesicles would lead to gradual transformation of medial cistern into a trans one. Another possibility is that recycling of resident Golgi proteins may occur not only by COP-coated, but also by other, possibly clathrin-coated, vesicles (which may contain Golgi enzymes, ref 13) or via different intermediates. Clearly, identification of the retrograde carrier (or carriers) for Golgi enzymes will be a key target in attempts to verify the maturation model.

Another key goal is dictated by the fact that the model is so far based purely on morphological evidence. Mostly because of this limitation, objections have been raised against the maturation model: 1) large polymeric cargoes could be disassembled into monomers, then be transported by conventional carriers and reassembled in the next cistern, and 2) polymer-containing cisterns might simply represent modified elements of the trans Golgi network. These objections have not been addressed directly. We believe that an animal cell secreting a large polymeric cargo that can be visualized by electron microscopy is molecularly and antigenically characterized and whose exit from the ER can be conveniently synchronized would provide the proper experimental system to address these questions. Embryonic collagen-secreting fibroblasts (ref 1 and references therein) appear to possess just the right properties, and so might greatly help verify the model. In a completely different approach, the maturation of one compartment (i.e., cis) into a trans compartment could, in principle, be followed directly by tagging cis and trans Golgi enzymes with green fluorescent proteins of different colors, then following by video microscopy the gradual acquisition of trans enzymes by the maturing cis compartment. This would, of course, involve the identification of a cell system where different Golgi compartments could be resolved by fluorescence microscopy.

Although the maturation model is consistent with a large fraction of the available evidence, we believe it is insufficient to explain all the experimental data. In particular, it does not account for two observations: 1) the presence of membrane continuities between the ER and the Golgi apparatus (see above) and between different Golgi compartments, sometimes forming a single interconnected membranous system (7, 9), and 2) the fact that different speeds of intra-Golgi transport have been reported for different cargoes. Large aggregates move through the Golgi complex more slowly than diffusable proteins [compare, for instance, the rate of traffic of collagen aggregates (14) with that of the G-protein of the vesicular stomatitis virus; ref 15], and not vice versa, as one might expect considering that small cargoes might be retarded by entering retrograde vesicles. Thus, the cistern maturation model and the lateral diffusion model are both compatible with only part of the existing data.

	THE SYNTHETIC MODEL

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The cistern maturation and lateral diffusion models are not mutually exclusive, and a schematic representation of how they might be combined is shown in Fig. 1. In this model, the transport of cargo occurs both tangentially and perpendicularly to the axis of the stack. Since a functional Golgi unit would consist of a cis-trans series of cisterns located in different stacks and joined by tubules or saccules (1), the diffusional transport of cargo should occur in a direction tangential, rather than perpendicular, to the stack. However, the retrograde movement of Golgi enzymes, presumably by vesicles, from distal (‘older’) cisterns to proximal (‘younger’) ones, would be expected to occur between closely opposed cisterns within a stack, resulting in the gradual maturation progression of cisterns toward the trans side in a direction perpendicular to the stack itself. The system operates precisely as a distillation tower (an analogy proposed previously by others; see ref 10), with cisterns functioning as plates, lateral diffusion of cargo as the rising vapor, and retrograde vesicles carrying Golgi enzymes (as well as escaped ER resident proteins or SNAREs returning to a proximal compartment; see ref 10) as the falling condensates. Anterograde traffic by vesicles has been studied and reviewed extensively (10), and is not discussed here. The key structural aspect of such a two-directional cargo movement is the spatial organization of the Golgi stacks as a single ribbon of interconnected stacks ( Fig. 1).

The synthetic model is consistent with the coexistence of vesicles, membranous connections between compartments, large intracisternal polymeric cargoes, and the observed lack of sharp separation between cis, medial, and trans-Golgi enzymes in different cisterns (13, 16). Moreover, it is compatible with the fact that intra-Golgi transport of SA appears to be slow (at least in animal cells) (14) compared with the fast intra-Golgi traffic of small conventional cargoes (15), since the two types of cargo move through different processes. The model can also accommodate the old observation (inconsistent with the ‘pure’ cistern maturation hypothesis) that transport can occur between the two separate Golgi complexes of heterokarions (17), if it is assumed that membranous bridges can be established between the two organelles by growth and fusion of tubular networks deriving from the two Golgis.

In conclusion, we believe that the model outlined here provides a novel realistic framework with which to interpret the morphological and functional evidence available today on the Golgi apparatus and to generate testable predictions. Some of the experiments suggested by the model are within reach of current technologies, and we hope they will increase our insight into the Golgi complexity.

	ACKNOWLEDGMENTS

 
We acknowledge support from the Italian CNR and AIRC.

	FOOTNOTES

 
¹ Correspondence: Mario Negri Sud Institute, Via Nazionale, 66030 S. Maria Imbaro, Chieti, Italy. E-mail: mironov{at}cmns.mnegri.it or luini{at}cmns.mnegri.it

² Abbreviations: COP, coat protein; ER, endoplasmic reticulum; SA, supramolecular aggregates.

Received for publication July 7, 1997. Accepted for publication October 23, 1997.

	REFERENCES

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