The F-BAR protein Syp1 negatively regulates WASp-Arp2/3 complex activity during endocytic patch formation.
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-2micro 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-2micro 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.
Boettner
Douglas R
DR
Department of Molecular and Cellular Pharmacology, Miller School of Medicine, University of Miami, Miami, FL 33136, USA.
D'Agostino
Jessica L
JL
Torres
Onaidy Teresa
OT
Daugherty-Clarke
Karen
K
Uygur
Aysu
A
Reider
Amanda
A
Wendland
Beverly
B
Lemmon
Sandra K
SK
Goode
Bruce L
BL
D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000483:Alleles; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D051316:Wiskott-Aldrich Syndrome Protein / Q000235:genetics / Q000378:metabolism; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000378:metabolism*
D051376:Actin-Related Protein 2-3 Complex; D000199:Actins; D002352:Carrier Proteins; C480361:LAS17 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C546997:Syp1 protein, S cerevisiae; D051316:Wiskott-Aldrich Syndrome Protein; D051300:Wiskott-Aldrich Syndrome Protein Family