A central role for the WH2 domain of Srv2/CAP in recharging actin monomers to drive actin turnover in vitro and in vivo.
Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K(d) approximately 1.5 microM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.
Chaudhry
Faisal
F
Department of Biology, Rosenstiel Basic Medical Science Research Center, Brandeis University, Waltham, Massachusetts, USA.
Little
Kristin
K
Talarico
Lou
L
Quintero-Monzon
Omar
O
Goode
Bruce L
BL
D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D003598:Cytoskeletal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004252:DNA Mutational Analysis; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D011817:Rabbits; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D003598:Cytoskeletal Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins