ARPC1/Arc40 mediates the interaction of the actin-related protein 2 and 3 complex with Wiskott-Aldrich syndrome protein family activators.
The actin-related protein 2 and 3 (Arp2/3) complex is a seven-subunit protein complex that nucleates actin filaments at the cell cortex. Despite extensive cross-linking, crystallography, genetic and biochemical studies, the contribution of each subunit to the activity of the complex remains largely unclear. In this study we characterized the function of the 40-kDa subunit, ARPC1/Arc40, of the yeast Arp2/3 complex. We showed that this subunit is indeed a stable component of the Arp2/3 complex, but its highly unusual electrophoretic mobility eluded detection in previous studies. Recombinant Arc40 bound the VCA domain of Wiskott-Aldrich syndrome protein family activators at a K(d) of 0.45 mum, close to that of the full complex with VCA (0.30 microm), and this interaction was dependent on the conserved tryptophan at the COOH terminus of VCA. Using a newly constructed Delta arc40 yeast strain, we showed that loss of Arc40 severely reduced the binding affinity of the Arp2/3 complex with VCA as well as the nucleation activity of the complex, suggesting that Arc40 contains an important contact site of the Arp2/3 complex with VCA. The Delta arc40 cells exhibited reduced growth rate, loss of actin patches, and accumulation of cables like actin aggregates, phenotypes typical of other subunit nulls, suggesting that Arc40 functions exclusively within the Arp2/3 complex.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Feng
F
Pan
Coumaran
C
Egile
Thomas
T
Lipkin
Rong
R
Li
D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051316:Wiskott-Aldrich Syndrome Protein
C497357:ARC40 protein, S cerevisiae; C477632:ARP2 protein, S cerevisiae; C102245:ARP3 protein, S cerevisiae; D051377:Actin-Related Protein 2; D051378:Actin-Related Protein 3; D000199:Actins; D008840:Microfilament Proteins; D021122:Protein Subunits; D011506:Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D051316:Wiskott-Aldrich Syndrome Protein