TY - JOUR
T1 - The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation
AU - Boettner, Douglas R.
AU - D'Agostino, Jessica L.
AU - Torres, Onaidy Teresa
AU - Daugherty-Clarke, Karen
AU - Uygur, Aysu
AU - Reider, Amanda
AU - Wendland, Beverly
AU - Lemmon, Sandra K.
AU - Goode, Bruce L.
PY - 2009/12/15
Y1 - 2009/12/15
N2 - Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results: We examined endocytosis in arp2-7 mutants by live-cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multicopy suppressors of arp2-7 and identified SYP1, an FCHO1 homolog, which contains F-BAR and AP-2μ homology domains. Overexpression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild-type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly in vitro. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains and depends on sequences in Las17/WASp outside of the VCA domain. Conclusions: Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.
AB - Background: Actin polymerization by Arp2/3 complex must be tightly regulated to promote clathrin-mediated endocytosis. Although many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. To address this, we analyzed the yeast arp2-7 allele, which is biochemically unique in causing unregulated actin assembly in vitro in the absence of Arp2/3 activators. Results: We examined endocytosis in arp2-7 mutants by live-cell imaging of Sla1-GFP, a coat marker, and Abp1-RFP, which marks the later actin phase of endocytosis. Sla1-GFP and Abp1-RFP lifetimes were accelerated in arp2-7 mutants, which is opposite to actin nucleation-impaired arp2 alleles or deletions of Arp2/3 activators. We performed a screen for multicopy suppressors of arp2-7 and identified SYP1, an FCHO1 homolog, which contains F-BAR and AP-2μ homology domains. Overexpression of SYP1 in arp2-7 cells slowed Sla1-GFP lifetimes closer to wild-type cells. Further, purified Syp1 directly inhibited Las17/WASp stimulation of Arp2/3 complex-mediated actin assembly in vitro. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains and depends on sequences in Las17/WASp outside of the VCA domain. Conclusions: Together, these data identify Syp1 as a novel negative regulator of WASp-Arp2/3 complex that helps choreograph the precise timing of actin assembly during endocytosis.
KW - CELLBIO
UR - https://www.scopus.com/pages/publications/71649086744
U2 - 10.1016/j.cub.2009.10.062
DO - 10.1016/j.cub.2009.10.062
M3 - Article
C2 - 19962315
AN - SCOPUS:71649086744
SN - 0960-9822
VL - 19
SP - 1979
EP - 1987
JO - Current Biology
JF - Current Biology
IS - 23
ER -