Browsing by Author "Schindler, Christina"

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Open Access
Analysis of SNAREs, Arf1p and regulators in intracellular transport
(2007) Schindler, Christina; Wolf, Dieter H. (Prof. Dr.)
Vesicular transport is an essential process allowing communication between different organelles in an eukaryotic cell. The small GTPase Arf1p regulates the generation of coated vesicles from donor organelles at many different levels of the secretory pathway. Arf1p cooperates with GTPase-activating proteins (GAPs) to hydrolyze the bound GTP, which subsequently induces the shedding of the proteinacious vesicle coat. Only then the vesicle is capable to undergo SNARE-mediated (soluble NSF attachment protein receptor) fusion with a target organelle to deliver its content. Previously, our lab showed that ArfGAPs can catalytically induce an altered conformation in vesicle SNARE proteins (v-SNAREs). The SNARE in the altered conformation is able to interact with Arf1p. Thus, the uptake of v-SNAREs in budding vesicles is facilitated. The current study extends the previous results to target membrane SNAREs (t-SNAREs) and shows that the ArfGAP-induced conformation enhances the formation of SNARE complexes. SNARE complex formation is an essential step during membrane fusion. Thus, the altered SNARE conformation is not only important during vesicle generation, but also for the consumption of transport vesicles. This let us speculate that ArfGAP proteins might act in a chaperone-like function on SNARE proteins. We were also able to show that SNARE proteins in the ArfGAP-induced SNARE conformation can interact with Sec17p and Sec18p, the yeast homologs of alpha-SNAP (soluble NSF attachment protein) and AAA-ATPase NSF (N-ethylmaleimide-sensitive factor). Both factors play a key role in membrane fusion by resolving cis-SNARE complexes. Data from this study indicate that the ATPase Sec18p has a second function during vesicle fusion as it can displace Arf1p from SNAREs. This process requires neither ATP-hydrolysis nor Sec18p´s co-factor Sec17p. Residual coat on the vesicle could still be required during an initial contact between the two fusing membranes. However, it would likely obstruct the final fusion event. Thus, Sec18p might be able to remove residual coat from a vesicle and allow final fusion to take place. After one round of vesicle fusion, v-SNAREs are recycled back to the donor compartment. Data presented in this study show that Snc1p and Snc2p, which are v-SNAREs involved in yeast exo- and endocytosis, interact physically and genetically with the ArfGAP Gcs1p, and that this interaction is essential for recycling of SNAREs via the trans-Golgi-Network (TGN) and endosomes. Furthermore, we were able to show that the COPI vesicle coat, which has been implicated in retrograde trafficking from the Golgi apparatus to the endoplasmic reticulum (ER) as well as in retrograde traffic within the Golgi apparatus, plays also an important role in the recycling of the post-Golgi SNAREs Snc1p and Snc2p. Besides Arf1p´s role in the formation of retrograde directed vesicles, Arf1p also participates in anterograde trafficking from the TGN, which is thought to be the main sorting station for anterograde cargo in an eukaryotic cell. In an attempt to identify new interactors of the small GTPase Arf1p, we found a novel fungi-specific protein family: the ChAPs (for Chs5p and Arf1p-interacting proteins). The ChAP family of proteins consists out of four members: Bch1p, Bch2p, Chs6p and Bud7p. These factors at least partially localize to the TGN, dependent on their interaction with Chs5p. Chs5p has been previously reported to be essential for the delivery of chitin-synthase III (Chs3p) to the yeast bud neck-region; a process Arf1p is also implicated in. We are able to show that Arf1p, Chs5p and individual members of the ChAP family interact genetically and physically. Arf1p, Chs5p and the ChAP proteins form high molecular complexes that contain the potential cargo Chs3p. Based on our findings, we suggest that the ChAP proteins are required for the transport of certain cargo in specialized transport vesicles. The ChAPs might function as cargo receptors, coat adaptors or even as novel coat. Altogether, this work has highlighted new interactors for the small GTPase Arf1p and new modes of function for its regulatory GAP proteins.