In the budding yeast Saccharomyces cerevisiae, endocytosis comprises bulk uptake (fluids and membranes) and receptor-mediated internalisation (membrane proteins). Both processes require efficient actin filament assembly. Key factors that nucleate the assembly of actin filaments are the Arp2/3 complex and a number of NPFs (Nucleation Promoting Factors), which are responsible for temporal and spatial regulation of Arp2/3 activity. In yeast, in addition to Las17p, the orthologue of WASP (Wiskott-Aldrich syndrome protein), one type I unconventional myosin (Myo5p) exhibits strong NPF activity through coordination with the WH2 (WASP Homology 2) domain containing protein Vrp1p, the yeast orthologue of WIP (WASP Interacting Protein). Here, we identified another key Vrp1p domain (Hof One Trap/HOT), which binds directly to the SH3 domain of the cytokinesis protein Hof1p, is important for Vrp1p function in vivo. The key function of the Vrp1p HOT domain is to counteract the inhibitory effect of the Hof1p SH3 domain in Myo5p-stimulated actin assembly and endocytosis. We have also revealed a novel actin monomer binding domain (VH2) in Vrp1p, which is functionally redundant with the WH2 domain. Receptormediated endocytosis requires stable interaction of Vrp1p with Las17p. However, we find that bulk uptake of fluid and membrane takes place without Vrp1p-Las17p association and requires only functional WH2 and HOT domains of Vrp1p. Finally, we identified a number of other potential Hof1p SH3 domain interactors using an affinity isolation approach and compared this interaction profile with those of several other yeast SH3 domains. The unique Vrp1p-Hof1p interaction pattern allows us to gain insight into the pathology of Wiskott-Aldrich syndrome.
| Identifer | oai:union.ndltd.org:ADTP/253018 |
| Creators | Ren, Gang |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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