Introduction Three kinds of transport vesicle have been functionally characterized at a molecular level and can be defined by both their membrane origin and their coat proteins (Kirchhausen, 2000). Clathrin-coated vesicles are formed from both the plasma membrane and the trans-Golgi network and mediate vesicular trafficking within the endosomal membrane system (Schmid, 1997). COPI-coated vesicles and COPII-coated vesicles are transport intermediates of the secretory pathway (Rothman and Wieland, 1996; Schekman and Orci, 1996; Barlowe, 1998). COPII vesicles emerge from the endoplasmic reticulum (ER) in order to export newly synthesized secretory proteins towards the Golgi (Schekman and Orci, 1996; Barlowe, 1998). COPI vesicles instead appear to be involved in both biosynthetic (anterograde) and retrograde transport within the Golgi complex (Orci et al., 1997), as well as mediating the recycling of proteins from the Golgi to the ER (Cosson and Letourneur, 1994; Letourneur et al., 1994; Sönnichsen et al., 1996). COPI vesicles have been used widely as a model system to study the molecular mechanism of transport vesicle biogenesis since they can be generated from purified Golgi membranes in vitro and isolated in appreciable amounts (Malhotra et al., 1989; Rothman, 1994). Here, we discuss the basic molecular components of COPI vesicles and their coordinated interplay in coat assembly and disassembly in the context of results obtained from various in vitro systems that reconstitute COPI vesicle biogenesis. Recruitment of COPI to Golgi membranes As depicted in Fig. 1, the coat structure of COPI vesicles consists of the heptameric coatomer protein complex, and the small GTPase ADP-ribosylation factor 1 (ARF1) (Rothman and Wieland, 1996). Coatomer and GDP-bound ARF1 (ARF1- GDP) are soluble cytosolic factors that are recruited to Golgi membranes in a GTP-dependent manner (Donaldson et al., 1992a; Palmer et al., 1993), and this initiates COPI-dependent vesicle budding (Rothman, 1994). GTP loading of ARF1 is catalyzed by a guanine-nucleotide-exchange factor (GEF) that is inhibited by the fungal metabolite brefeldin A (BFA) (Donaldson et al., 1992b; Helms and Rothman, 1992). BFA causes a redistribution of COPI components to cytoplasm, a breakdown of COPI vesicle formation and a redistribution of Golgi enzymes into the ER (Lippincott-Schwartz et al., 1989; Donaldson et al., 1990; Orci et al., 1991). Several ARF-specific GEFs have been described at the molecular level and can be classified as BFA resistant or BFA sensitive (Jackson and Casanova, 2000). Currently, which of the BFA-sensitive ARF- GEFs promotes COPI vesicle formation is not clear. Nucleotide exchange on ARF1 has widely been regarded to be the initial step of COPI coat assembly (Rothman, 1994). However, recent data demonstrate that ARF1-GDP interacts with the Golgi in a specific manner (Gommel et al., 2001) and that this interaction precedes nucleotide exchange. On the basis of crosslinking studies as well as ARF1-binding experiments employing native Golgi membranes, p23, a type I transmembrane protein known to play a key role in COPI coat assembly (Sohn et al., 1996; Bremser et al., 1999) was identified as an ARF1-GDP receptor (Gommel et al., 2001). p23 belongs to the p24 protein family, members of which share common structural features such as double lysine or double arginine residues in their cytoplasmic tails (for a review, see Emery et al., 1999). While the first member was identified in 1991 (Wada et al., 1991), the existence of a family of related proteins was reported in 1995 by Rothman and colleagues (Stamnes et al., 1995). Currently, six family members have been identified in higher eukaryotes, whereas eight are known in yeast (Emery et al., 1999). The identification of p23 as an ARF-GDP receptor (Gommel et al., 2001) is consistent with recent in vivo studies demonstrating energy transfer between p23-CFP and ARF1-YFP in living cells, an interaction detected only under conditions that allow ARF-mediated GTP hydrolysis (Majoul et al., 2001). Peptide-mapping studies 3235 Vesicular transport is the predominant mechanism for exchange of proteins and lipids between membrane-bound organelles in eukaryotic cells. Golgi-derived COPI-coated vesicles are involved in several vesicular transport steps, including bidirectional transport within the Golgi and recycling to the ER. Recent work has shed light on the mechanism of COPI vesicle biogenesis, in particular the machinery required for vesicle formation. The new findings have allowed us to generate a model that covers the cycle of coat recruitment, coat polymerization, vesicle budding and uncoating. Key words: Coatomer, ARF, p24 proteins, Vesicular transport, Coat assembly, Protein secretion, Golgi Summary Vesicular transport: the core machinery of COPI recruitment and budding Walter Nickel*, Britta Brügger and Felix T. Wieland Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany *Author for correspondence (e-mail: walter.nickel@urz.uni-heidelberg.de) Journal of Cell Science 115, 3235-3240 (2002) © The Company of Biologists Ltd Commentary