Negative regulation of yeast WASp by two SH3 domain-containing proteins.
BACKGROUND
WASp family proteins promote actin filament assembly by activating Arp2/3 complex and are regulated spatially and temporally to assemble specialized actin structures used in diverse cellular processes. Some WASp family members are autoinhibited until bound by activating ligands; however, regulation of the budding yeast WASp homolog (Las17/Bee1) has not yet been explored.


RESULTS
We isolated full-length Las17 and characterized its biochemical activities on yeast Arp2/3 complex. Purified Las17 was not autoinhibited; in this respect, it is more similar to SCAR/WAVE than to WASp proteins. Las17 was a much stronger activator of Arp2/3 complex than its carboxyl-terminal (WA) fragment. In addition, actin polymerization stimulated by Las17-Arp2/3 was much less sensitive to the inhibitory effects of profilin compared to polymerization stimulated by WA-Arp2/3. Two SH3 domain-containing binding partners of Las17, Sla1 and Bbc1, were purified and were shown to cooperate in inhibiting Las17 activity. The two SLA1 SH3 domains required for this inhibitory activity in vitro were also required in vivo, in combination with BBC1, for cell viability and normal actin organization.


CONCLUSIONS
Full-length Las17 is not autoinhibited and activates Arp2/3 complex more strongly than its WA domain alone, revealing an important role for the Las17 amino terminus in Arp2/3 complex activation. Two of the SH3 domain-containing ligands of Las17, Sla1 and Bbc1, cooperate to inhibit Las17 activity in vitro and are required for a shared function in actin organization in vivo. Our results show that, like SCAR/WAVE, WASp proteins can be controlled by negative regulation through the combined actions of multiple ligands.
Rodal
Avital A
AA
Department of Molecular and Cell Biology, University of California, Berkeley, 94720, USA.
Manning
Amity L
AL
Goode
Bruce L
BL
Drubin
David G
DG
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008840:Microfilament Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism*; D051304:Profilins; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism*; D014170:Transformation, Genetic; D051316:Wiskott-Aldrich Syndrome Protein
C477632:ARP2 protein, S cerevisiae; C102245:ARP3 protein, S cerevisiae; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D002352:Carrier Proteins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; C480361:LAS17 protein, S cerevisiae; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D051304:Profilins; C082265:SLA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein