Three Ways to Make a Vesicle

REVIEWS THREE WAYS TO MAKE A VESICLE Tomas Kirchhausen Cargo molecules have to be included in carrier vesicles of different forms and sizes to be transported between organelles. During this process, a limited set of proteins, including the coat proteins COPI, COPII and clathrin, carries out a programmed set of sequential interactions that lead to the budding of vesicles. A general model to explain the formation of coated vesicles is starting to emerge but the picture is more complex than we had imagined. ENDOCYTIC PATHWAY Transport of proteins and lipids along the ENDOCYTIC or energy dependent and includes sorting of cargo to the Macromolecules are taken up SECRETORY PATHWAYS is a hallmark of eukaryotic cells. The forming coat. Coat propagation, the second step in the by invagination of the plasma membrane fluxes along these pathways are very large process, couples further addition of coat components membrane. They first arrive in and rapid. A FIBROBLAST kept in resting conditions in a and additional recruitment of cargo with invagination of early endosomes, then late endosomes, and finally tissue culture plate internalizes an amount of mem- the underlying membrane. When formation of the coat lysosomes, where they are brane equivalent to the whole surface area of the cell in ends, the vesicle buds by scission of the neck connecting degraded by hydrolases. one hour. Inside the cell, it often takes only seconds for a the deeply invaginated membrane to the donor surface. carrier vesicle to move from the donor membrane to This is a relatively simple step for COPI- and COPII- SECRETORY PATHWAY the acceptor organelle. But intracellular traffic is not coated vesicles but involves a significant amount of regu- Secretory or membrane proteins are inserted into the only rapid, it is also very selective. Only a subset of the lation for clathrin-coated vesicles. Finally, during uncoat- endoplasmic reticulum. They proteins and lipids in the donor membrane are allowed ing, the coat components are released so that membrane are then transported through into the transport vesicle, effectively preventing the fusion can occur between the naked vesicle and the target the Golgi to the trans-Golgi homogenization of membrane components and per- organelle. This article discusses the emerging molecular network, where they are sorted to their final destination. mitting membranous organelles to maintain distinct rationale for coated-vesicle assembly for, in order of identities throughout the life of the cell. increasing complexity, COPII, COPI and clathrin. FIBROBLAST Considerable progress has been made towards under- Common cell type found in standing the molecular basis of membrane traffic1–8.A Pathways of membrane traffic connective tissue in many parts number of traffic pathways have been defined, their COPI and COPII vesicles traffic between the endoplas- of the body, which secretes an extracellular matrix rich in major protein components identified and the structures mic reticulum (ER) and the Golgi complex — COPI collagen and other of several of the key components determined at atomic primarily from the Golgi to the ER and between Golgi macromolecules and connects or molecular resolution. The best studied traffic path- cisternae, and COPII from the ER to the Golgi (FIG. 1). cell layers. ways are those that use carrier vesicles that are clearly The clathrin pathway has two major routes, from the MACROPINOCYTOSIS identifiable by their coats, made of the coatomer COPI, plasma membrane to the early endosome and from the Actin-dependent process by of COPII, or of clathrin and its partners (FIG. 1).During Golgi to the endosome. Other structures have been which cells engulf large volumes the formation of a vesicle, a limited set of coat proteins observed in the cell that do not have COP or clathrin of fluids. (TABLE 1) carries out a programmed set of sequential coats. Internalization from the plasma membrane can interactions that lead to budding from the parent mem- also occur via MACROPINOCYTOSIS, PHAGOCYTOSIS and proba- brane, uncoating, fusion with a target membrane and bly through CAVEOLAE9. In the secretory pathway, poorly recycling of the coat components. There are clear similar- understood tube-like structures connect the Golgi with Harvard Medical School, ities and differences between the ways that COPI, COPII the plasma membrane and the ER10–13. In the endocytic 200 Longwood Avenue, and clathrin coats handle these steps (TABLE 2). pathway, tubules emerge from early endosomes and Boston, Massachusetts 14 02115, USA. e-mail: The first group of reactions, leading to the specific participate in recycling to the plasma membrane . kirchhausen@crystal. recruitment of coat components to the corresponding These carrier structures have been hard to study, partly harvard.edu donor membrane, form the initiation step. This step is because they tend to be heterogeneous. In some cases, NATURE REVIEWS | MOLECULAR CELL BIOLOGY © 2000 Macmillan Magazines Ltd VOLUME 1 | DECEMBER 2000 | 187 REVIEWS molecular detail. Nevertheless, there is good agreement Clathrin coats in the field that the major components have been identi- Recycling endosome fied and there is a good working model for the sequence in which they act (FIG. 2). Biochemical and cell biological observations. Although several as-yet-undefined membrane components are Early endosome Late endosome presumably necessary for efficient operation of the COPII pathway, vesicular transport can be minimally Clathrin coats reconstituted using three cytosolic components contain- ing a total of five proteins: the Sec23p–Sec24p complex, the Sec13p–Sec31p complex and the small GTPase Trans-Golgi Sar1p (REF.16). These proteins support a cargo-carrying budding reaction from isolated ER membranes. Deletion of the genes for most of these components is COPII coats Cis-Golgi lethal for yeast, highlighting the strict dependence on COPI coats this pathway for ER-to-Golgi traffic. Lysosome The events driven by these five cytosolic proteins, Endoplasmic reticulum (ER) together with the membrane components required for targeting and fusion, are varied and complex (TABLE 2). Cargo capture, deformation of the budding membrane, scission to detach the forming vesicle from the donor membrane and coat release before fusion of the vesicle with the Golgi network all occur in this minimal system. Figure 1 | The major membrane traffic pathways that use carrier vesicles coated with The GTP-binding protein Sar1p is particularly impor- COPI, COPII and clathrin in eukaryotic cells. In the biosynthetic pathway, newly tant for the budding reaction because its activation initi- synthesized molecules are transported from the endoplasmic reticulum to the Golgi and from ates coat formation; it also recruits part of the ‘label’ one cisterna of the Golgi to the next until they reach the trans-Golgi network. There, sorting needed for correct vesicle targeting and fusion. The occurs, directing traffic to the plasma membrane or to endosomes. In the endocytic pathway, GDP-bound form of Sar1p is normally cytosolic and is macromolecules are internalized at the plasma membrane and forwarded to early endosomes, from where they are either recycled to the plasma membrane through recycling endosomes or recruited to the ER membrane upon interaction with forwarded towards degradation in late endosomes and lysosomes. Sec12p, an ER-bound membrane protein that serves as the guanine exchange factor (GEF) for Sar1p (REF.17). Sar1p–GTP then facilitates the association of the they might be used for large-scale movement of selec- Sec23p–Sec24p complex with cargo proteins. tively captured membrane components12,15. The most The Sec23p–Sec24p complex is probably the com- important distinction between coated vesicles and other ponent responsible for cargo recognition18,19 but the forms of membrane carriers is the presence of an iden- sorting signals recognized by the complex remain to be tifiable protein coat, which assembles at a particular identified. Members of the p24 family of transmem- region of the membrane, locally deforming it. Although brane proteins bind to Sec23p through a cytosolic the events required are similar — tubes, diphenylalanine motif. As these proteins are required macropinosomes and phagosomes also need to form, for efficient ER-to-Golgi traffic of some cargo pinch off, reach their target and fuse with it just like proteins20, it is thought that they might serve as cargo coated vesicles — it is impossible to transfer our under- adaptors21. In addition to recruiting the Sec23p–Sec24p standing of vesicle traffic to these other mechanisms. complex, the GTP-bound form of Sar1p activates Sec23p to bind SNARE proteins involved in the speci- COPII ficity of targeting and in the fusion reaction of vesicles COPII components, and COPII-coated vesicles, were with acceptor membranes18. ER membranes with originally discovered in the yeast Saccharomyces cerevisiae Sec23p–Sec24p and Sar1p can then recruit using genetic approaches coupled with a cell-free assay Sec13p–Sec31p (REF.16). The complex is likely to act as a that measured the transfer of a marker protein from the scaffold, very much like clathrin, to drive membrane 3,8 PHAGOCYTOSIS ER to the Golgi . This pathway has a mammalian coun- deformation and to complete vesicle budding. Actin-dependent process, by terpart and most of the COPII components have been Completing the cycle, Sec23p acts as a GTPase-activat- which cells engulf external identified (TABLE 1). Newly synthesized proteins destined ing protein (GAP) for Sar1p. It is thought that, after particulate material by for secretion are sorted into COPII-coated vesicles at spe- GTP hydrolysis, Sar1p–GDP is released, leading to extension and fusion of pseudopods around each cialized regions of the ER, which in mammalian cells do uncoating before fusion of the vesicle to the target particle. not contain membrane-bound ribosomes. membrane and formation of a new coated vesicle. GTP Biochemical analysis has led to a relatively detailed hydrolysis by Sar1p is thus a timer, triggering uncoating CAVEOLA model for the mechanism of COPII vesicle formation. at a suitable interval after coat formation.

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