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Abstract
Actin nucleation is the key rate limiting step in the process of actin polymerization, and tight regulation of this process is critical to ensure actin filaments form only at specific times and at defined regions of the cell. Arp2/3 is a well-characterised protein complex that can promote nucleation of new filaments, though its activity requires additional nucleation promotion factors (NPFs). The best recognized of these factors are the WASP family of proteins that contain binding motifs for both monomeric actin and for Arp2/3. Previously we demonstrated that the yeast WASP homologue, Las17, in addition to activating Arp2/3 can also nucleate actin filaments de novo, independently of Arp2/3. This activity is dependent on its polyproline rich region. Through biochemical and in vivo analysis we have now identified key motifs within the polyproline region that are required for nucleation and elongation of actin filaments, and have addressed the role of the WH2 domain in the context of actin nucleation without Arp2/3. We have also demonstrated that full length Las17 is able to bind liposomes giving rise to the possibility of direct linkage of nascent actin filaments to specific membrane sites to which Las17 has been recruited. Overall, we propose that Las17 functions as the key initiator of de novo actin filament formation at endocytic sites by nucleating, elongating and tethering nascent filaments which then serve as a platform for Arp2/3 recruitment and function.
Highlights
Las17 is the primary activator of Arp2/3-driven actin nucleation in the budding yeast Saccharomyces cerevisiae, and is required for membrane invagination during endocytosis [1,2,3]
Given that profilin does not contribute to the Las17 actin polymerization mechanism we considered intrinsic properties of the region itself and whether the role of the C-terminal polyproline tracts (C-PP) might be tightly coupled to the nucleation role of the basic residues in the N-terminal portion of the region
Mechanisms governing nucleation of actin filaments de novo are critical in order to allow filaments to form at appropriate sites in cells
Summary
Las is the primary activator of Arp2/3-driven actin nucleation in the budding yeast Saccharomyces cerevisiae, and is required for membrane invagination during endocytosis [1,2,3]. The WASP family of proteins, which include WASP, N-WASP, WAVE, and WASH have been studied extensively as activators of Arp2/3-driven actin polymerization [4,5,6,7]. Las has a similar domain structure to mammalian WASP having an N-terminal WH1 domain, a central proline rich region and a C-terminal WCA region. In both Las and WASP, actin nucleation activity is attributed to this C-terminal region that contains. Arp2/3-Independent Actin Nucleation by Las17/WASP data collection and analysis, decision to publish, or preparation of the manuscript
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