Vesicle formation can be dissected into three stages (Nickel and Wieland, 1997; Wieland and Harter, 1999). First, a member o f the Ras superfamily o f GTPases, Sarpl for COPII or A R Fl (ADP-ribosylation factor) for COPI, is recruited to the donor membrane from the cytosol by the catalysed exchange o f GDP for GTP. Second, is the formation o f a vesicle bud that occurs concomitant with the GTPase-mediated recruitment o f hetero-oligomeric coat protein complexes to the donor membrane, and thirdly a periplasmic fusion takes place to release a coated vesicle from the donor compartment.
The reconstitution o f vesicle formation in vitro allowed the identification o f the niinimal machinery for COPI- and COPII-vesicle formation. The formation o f COPII- vesicles requires Sarlp, the Secl3p complex and the Sec23p complex (Barlowe et al., 1994), and formation o f COPI-vesicles requires coatomer and A R Fl (Ostermann et al., 1993). Vesicle formation is initiated by the recruitment o f S arlp (COPII-vesicles) or ARFl (COPI-vesicles) from the cytosol to the membrane. These GTPases exist in the GDP-bound form in the cytosol and exchange their GDP for GTP upon recruitment to the membrane. The nucleotide exchange is catalysed by guanine nucleotide exchange factors (GEFs) and induces a conformational change in the exposing N-terminal myristoyl group which allows its attachment to the membrane (Goldberg, 1998). Activation o f the small GTPases is followed by coat protein recruitment.
In the case o f COPII, the Sec23p complex binds, followed by the Secl3p complex. A complex comprising o f Sar 1 p-Sec 16p-Sed4 (Gimeno et al., 1995) serves as a docking pad for the Sec23p complex (Espenshade et al., 1995) and thus ensures that the correct coat complex is recruited to the correct donor membrane. A direct interaction o f the Sec23p complex with SNAREs (e.g. B etlp , B oslp , Sed5p), cargo receptors and cargo (e.g. p24, ERGIC53, VSVG, M annll, NAGTI) ensures that the appropriate cargo molecules are selectively incorporated into nascent vesicles (Springer et al., 1999).
Chapter 1______________________________________________________ Introduction
Furthermore, essential v-SNAREs (e.g. Sec22p) are selectively incorporated into the vesicles by binding to the coat-binding SNAREs, thus ensuring that vesicles are not formed unless they contain all the necessary components required for later steps in the transport pathway (Springer et al., 1999). Sec24p also serves as a binding site for the Secl3p complex. Subsequent polymerisation o f multiple Sec23p/Secl3p complexes further clusters cargo molecules by crosslinking the Sec23p complexes and this induces bud formation.
In case the o f COPI-vesicle formation, coatomer (a complex o f seven distinct subunits or COPs) binds to the membrane en bloc (Orci et al., 1993) which entails a direct interaction between ARF-GTP and the p-COP subunit (Zhao et al., 1997). The assembly o f the COPI-coat induces a deformation o f the donor membrane into a coated bud which is sufficient to generate COPI-vesicles from acidic liposomes (Spang and Schekman, 1998). ARF-GTP also stimulates phospholipase D (PLD), which hydrolyses phosphatidylcholine (PC) to phosphatidic acid (PA) and choline (Brown et al., 1993). This change in the lipid composition has been shown to enhance coatomer binding to the membrane and thus may contribute to the formation o f COPI-vesicles (Ktistakis et al., 1996). y-COP interacts directly with the KKXX-motif, which is found on the cytoplasmic tails o f escaped ER residents and the p24 family o f proteins (Harter and Wieland, 1998). Interestingly, members o f the p24 family have a propensity to form large hetero-oligomers (Fullekrug et al., 1999), and so may mark sites from which COPI-vesicles are destined to bud, thus providing a mechanism to ensure that cargo enters COPI-vesicles.
Once a coated bud has formed it must be pinched off the membrane to generate a free vesicle. Fission o f COPI-vesicles from Golgi membranes requires palmitoyl-CoA as an additional factor (Ostermann et al., 1993), whereas no additional factors are required to release COPII-vesicles (M atsuoka et al., 1998). Interestingly, a protein called BARS (BFA-induced ADP-ribosylated substrate) was recently identified to cause non specific fission o f Golgi membranes by transferring a range o f fatty acids (including palmitate) from CoA to lysophosphatidic acid (LPA) to generate PA, a cone shaped
Chapter 1______________________________________________________ Introduction
lipid, that induces a negative membrane curvature and subsequent membrane fission (Weigert et al., 1999). A similar mode o f action was proposed for endothilin-I, a protein that interacts with the GTP-driven garrotte dynamin (Stowell et al., 1999), to drive synaptic-like microvesicle or clathrin-coated vesicle formation (Schmidt et al., 1999). The reason that clathrin-coated vesicles have a more elaborate fission machinery than COPI-vesicles and COPII-vesicles may reflect the biophysical properties o f the membranes from which they form (Bednarek et al., 1996). The higher cholesterol content at the Golgi and plasma membrane compared to the ER may confer greater membrane rigidity and make coated vesicle formation a more energy demanding process, thus requiring a specialised fission machinery.
GTP-hydrolysis by Sar Ip/A R Fl causes their release from the membrane and subsequent uncoating o f the COPII- or COPI-vesicle (Barlowe et al., 1994; Tanigawa et al., 1993). Since both Sarlp and A R Fl hydroylse GTP at a slow rate, a GTPase activating protein (GAP) is thought to stimulate their GTPase activity. This is a component o f the COPII-coat in the case o f S arlp (Yoshihisa et al., 1993) and a protein that binds to the KDEL-receptor in the case o f A R Fl (Aoe et al., 1997). Vesicle uncoating is probably required to reveal the v-SNARE machinery, an essential component for the subsequent docking o f the vesicle with its acceptor compartment.