Mylène Amherdt

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Brefeldin A's effects on endosomes, lysosomes, and the TGN suggest a general mechanism for regulating organelle structure and membrane traffic.

Addition of brefeldin A (BFA) to most cells results in both the formation of extensive, uncoated membrane tubules through which Golgi components redistribute into the ER and the failure to transport molecules out of this mixed ER/Golgi system. In this study we provide evidence that suggests BFAs effects are not limited to the Golgi apparatus but are reiterated throughout the central vacuolar system. Addition of BFA to cells resulted in the tubulation of the endosomal system, the trans-Golgi network (TGN), and lysosomes. Tubule formation of these organelles was specific to BFA, shared near identical pharmacologic characteristics as Golgi tubules and resulted in targeted membrane fusion. Analogous to the mixing of the Golgi with the ER during BFA treatment, the TGN mixed with the recycling endosomal system. This mixed system remained functional with normal cycling between plasma membrane and endosomes, but traffic between endosomes and lysosomes was impaired.

COPII-coated vesicle formation reconstituted with purified coat proteins and chemically defined liposomes.

COPII vesicle formation requires only three coat assembly subunits: Sar1p, Sec13/31p, and Sec23/24p. PI 4-phosphate or PI 4,5-bisphosphate is required for the binding of these proteins to liposomes. The GTP-bound form of Sar1p recruits Sec23/24p to the liposomes as well as to the ER membranes, and this Sar1p-Sec23/24p complex is required for the binding of Sec13/31p. Ultrastructural analysis shows that the binding of COPII coat proteins to liposomes results in coated patches, coated buds, and coated vesicles of 50-90 nm in diameter. Budding proceeds without rupture of the donor liposome or vesicle product. These observations suggest that the assembly of the COPII coat on the ER occurs by a sequential binding of coat proteins to specific lipids and that this assembly promotes the budding of COPII-coated vesicles.

Bidirectional Transport by Distinct Populations of COPI-Coated Vesicles

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

BREFELDIN A, A DRUG THAT BLOCKS SECRETION, PREVENTS THE ASSEMBLY OF NON-CLATHRIN-COATED BUDS ON GOLGI CISTERNAE

We report that brefeldin A prevents the assembly of non-clathrin-coated vesicles from Golgi cisternae in a cell-free system. This finding provides a simple molecular explanation for the primary effect of this remarkable compound in blocking constitutive secretion. We further report that when coated vesicle assembly is blocked, extensive tubule networks form that connect previously separate cisternae and stacks into a single topological unit, allowing the intermixing of contents of Golgi cisternae, presumably by lateral diffusion. Formation of the tubule networks requires ATP, cytosol, and the general fusion protein NSF. Tubule networks may be related to the membrane tubules mediating retrograde transport in vivo.

COUPLING OF COAT ASSEMBLY AND VESICLE BUDDING TO PACKAGING OF PUTATIVE CARGO RECEPTORS

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

Stepwise assembly of functionally active transport vesicles.

Budding of COP-coated vesicles (the likely carriers of newly synthesized proteins from the endoplasmic reticulum through the Golgi stack) from Golgi cisternae requires ADP-ribosylation factor (ARF), coatomer proteins from the cytosol, GTP, and fatty acyl-coenzyme A (CoA). The assembly of coated buds on the membranes requires coatomer, ARF, and GTP. When palmitoyl-CoA is added, membrane fission occurs at the coated bud, releasing coated vesicles. We show that COP-coated vesicles can be generated stepwise in vitro and isolated in a functionally active form, demonstrating that the minimal set of cytosolic components required for their formation as well as principal steps in their assembly have been identified.

COPI- and COPII-coated vesicles bud directly from the endoplasmic reticulum in yeast

The cytosolic yeast proteins Sec13p-Sec31p, Sec23p-Sec24p, and the small GTP-binding protein Sar1p generate protein transport vesicles by forming the membrane coat termed COPII. We demonstrate by thin section and immunoelectron microscopy that purified COPII components form transport vesicles directly from the outer membrane of isolated yeast nuclei. Another set of yeast cytosolic proteins, coatomer and Arf1p (COPI), also form coated buds and vesicles from the nuclear envelope. Formation of COPI-coated, but not COPII-coated, buds and vesicles on the nuclear envelope is inhibited by the fungal metabolite brefeldin A. The two vesicle populations are distinct. However, both vesicle types are devoid of endoplasmic reticulum (ER) resident proteins, and each contains targeting proteins necessary for docking at the Golgi complex. Our data suggest that COPI and COPII mediate separate vesicular transport pathways from the ER.

The trans-most cisternae of the Golgi complex: A compartment for sorting of secretory and plasma membrane proteins

The intracellular site for the sorting of proteins destined for regulated or constitutive pathways is presently unknown for any one cell. By immunoelectron microscopy, we directly followed the routes taken by a regulated hormone, insulin, and a constitutive protein, hemagglutinin. Both proteins are present in individual Golgi stacks where they appear randomly distributed throughout the cisternae. In contrast, the two proteins do not colocalize outside the Golgi area:insulin is concentrated in dense-core secretory granules, while hemagglutinin is found predominantly in clear 100-300 nm vesicles. These vesicles do not label significantly with an endocytic tracer, indicating that they are exocytic carriers for hemagglutinin. The site at which the two proteins diverge is the clathrin-coated, trans-most cisterna of the Golgi, where the packaging of proinsulin takes place.

Direct identification of prohormone conversion site in insulin-secreting cells

We have localized proinsulin in B cells of human and rat pancreatic islets, using a proinsulin-specific monoclonal antibody revealed by immunocytochemistry. Proinsulin is abundant in Golgi stacks and clathrin-coated secretory granules. It rapidly disappears from these compartments when protein synthesis is inhibited. Depletion of ATP stores prevents movement of proinsulin from the Golgi stacks to the secretory granules; under these conditions, the prohormone in preformed coated granules is converted to insulin, whereas that bound to the Golgi complex is not. Non-coated granules show a low level of proinsulin reactivity under all incubation protocols. These findings provide direct evidence that coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion. They also suggest that the Golgi stack is not involved in conversion, and that intercisternal transport and coated granule formation are hitherto unrecognized energy-requiring steps that precede conversion.