A high-affinity interaction with ADP-actin monomers underlies the mechanism and in vivo function of Srv2/cyclase-associated protein.
Cyclase-associated protein (CAP), also called Srv2 in Saccharomyces cerevisiae, is a conserved actin monomer-binding protein that promotes cofilin-dependent actin turnover in vitro and in vivo. However, little is known about the mechanism underlying this function. Here, we show that S. cerevisiae CAP binds with strong preference to ADP-G-actin (Kd 0.02 microM) compared with ATP-G-actin (Kd 1.9 microM) and competes directly with cofilin for binding ADP-G-actin. Further, CAP blocks actin monomer addition specifically to barbed ends of filaments, in contrast to profilin, which blocks monomer addition to pointed ends of filaments. The actin-binding domain of CAP is more extensive than previously suggested and includes a recently solved beta-sheet structure in the C-terminus of CAP and adjacent sequences. Using site-directed mutagenesis, we define evolutionarily conserved residues that mediate binding to ADP-G-actin and demonstrate that these activities are required for CAP function in vivo in directing actin organization and polarized cell growth. Together, our data suggest that in vivo CAP competes with cofilin for binding ADP-actin monomers, allows rapid nucleotide exchange to occur on actin, and then because of its 100-fold weaker binding affinity for ATP-actin compared with ADP-actin, allows other cellular factors such as profilin to take the handoff of ATP-actin and facilitate barbed end assembly.
Mattila
Pieta K
PK
Program in Cellular Biotechnology, Institute of Biotechnology, University of Helsinki, Helsinki 00014, Finland.
Quintero-Monzon
Omar
O
Kugler
Jamie
J
Moseley
James B
JB
Almo
Steven C
SC
Lappalainen
Pekka
P
Goode
Bruce L
BL
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000244:Adenosine Diphosphate / Q000737:chemistry*; D000483:Alleles; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002942:Circular Dichroism; D003598:Cytoskeletal Proteins / Q000378:metabolism / Q000502:physiology*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008840:Microfilament Proteins / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013997:Time Factors
D051339:Actin Depolymerizing Factors; D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D005982:Glutathione Transferase