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Abstract

In Eukaryotes vesicular transport mechanisms collectively ensure the transport and distribution of proteins and lipids between cellular compartments to maintain their unique composition and their specialized functions. COPI vesicles mediate retrograde transport from the ERGIC/Golgi to the ER as well as intra-Golgi transport. Formation as well as consumption of COPI-coated transport vesicles is directly controlled by small GTPases of the Arf family, which in turn are regulated via specific Guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs). COPI vesicle formation is initiated by recruitment of Arf1 to membranes, which subsequently recruits the heptameric coat complex coatomer. GTP hydrolysis within Arf1 is a prerequisite for COPI vesicle uncoating. Three ArfGAPs are associated with COPI vesicle formation in mammalian cells: ArfGAP1, ArfGAP2 and ArfGAP3. During the course of this work mechanistic aspects of COPI vesicle biogenesis were investigated: i) the interaction of ArfGAPs with coatomer isoforms, ii) the regulation of ArfGAP activity by p24 family proteins, and iii) the molecular mechanism of COPI vesicle uncoating. We have determined the dissociation constants of the ArfGAPs for each of the four individual coatomer isoforms and found that all three ArfGAPs displayed a higher affinity for the γ1ζ1 isoform than for the other isoforms. This result is in accordance with the localization of both ArfGAP2/3 and γ1ζ1 to the cis-Golgi, whereas ArfGAP1 is equally distributed throughout the Golgi apparatus. Furthermore, we have investigated an effect on the GAP activity of ArfGAP1 and ArfGAP2 of the cytoplasmic tail of the transmembrane protein p23, a COPI vesicle machinery protein. p23 was reported to induce a conformational change in coatomer, resulting in a structure that resembles coatomer conformation within the polymerized COPI coat. Interestingly, p23 influenced the activity of ArfGAP1 and ArfGAP2 in an opposite fashion: whereas ArfGAP2 displayed a higher rate of Arf1 GTP hydrolysis in the presence of p23, ArfGAP1 displayed a lower rate. Thus, ArfGAP2/3 might preferentially interact with polymerized coatomer as found on a completed COPI vesicle. Although GTP hydrolysis in Arf1 is commonly considered necessary for coat disassembly, it remains obscure whether it is sufficient to complete this process. To investigate this pivotal mechanistic question, we have established a real-time assay to follow the fate of the COPI coat components of purified vesicles upon addition of ArfGAPs, and discovered an unanticipated essential role of the non-catalytic domains of ArfGAPs. While GTP-hydrolysis within Arf1, induced by the isolated catalytic domain of the ArfGAP, released the small GTPase from the coat, the network of coatomer remained associated with vesicle membranes. Only in the presence of full-length ArfGAP1, including the non-catalytic part, the coat network was completely disassembled. We propose that the energy released upon GTP-hydrolysis in Arf1 is coupled by GAP-coatomer interactions to mediate conformational changes in coatomer that are required for COPI coat disassembly.