The F-BAR Protein Syp1 Negatively Regulates WASp-Arp2/3 Complex Activity during Endocytic Patch Formation

Abstract

Background Actin polymerization by Arp2/3 complex must be tightly regulated to promote endocytosis. While many Arp2/3 complex activators have been identified, mechanisms for its negative regulation have remained more elusive. 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 multi-copy suppressors of arp2-7 and identified SYP1, a FCHO1 homologue, which contains F-BAR and AP-2μ homology domains. Over-expression 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. This activity was mapped to a fragment of Syp1 located between its F-BAR and AP-2μ homology domains, and depends on non-VCA sequences in Las17/WASp. 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.

Role of Scd5, a protein phosphatase-1 targeting protein, in phosphoregulation of Sla1 during endocytosis

“…Las17, the yeast WASp homolog is a major actin nucleation promoting factor (NPF), but it arrives around the time of the late coat factors in wild-type cells and is subject to inhibition by Sla1 and Syp1 during the immobile phase (Boettner et al, 2009;Kaksonen et al, 2003;Rodal et al, 2003;Sun et al, 2006). In the scd5 mutant Las17-GFP had a delay of about +14 seconds, which was fairly similar to that of Sla1 ( Fig.…”

“…Las17 (yeast WASp) arrives at the cortex during the immobile phase of internalization , but its NPF activity is inhibited by Sla1 and Syp1 during this period (Boettner et al, 2012;Boettner et al, 2009;Rodal et al, 2003). We found that overexpression of LAS17 suppressed the ts growth of sla1-DSR at 36˚C and partially rescued the Pan1 lifetimes from ,260 s to ,165 s, P,0.0001.…”

“…Other coat module factors include the clathrin adaptors Ent1/2 (epsins) and Yap1801/2, which also bind EH proteins like Pan1 (Aguilar et al, 2003;Wendland and Emr, 1998). Syp1 and Ede1 leave the membrane just prior to the rapid mobile actin phase (Boettner et al, 2009;Stimpson et al, 2009;Toshima et al, 2006), which is associated with recruitment of several actin-remodeling factors (e.g. Arp2/3 complex, capping protein, type 1 myosins, Abp1, etc.).…”

“…2) (for reviews, see Boettner et al, 2012;Weinberg and Drubin, 2012). Clathrin, Ede1 [an Eps15 homology (EH) domain protein] and Syp1 (FCHO1/2 homolog) arrive at the endocytic site up to 1-2 min before the arrival of other coat factors (Boettner et al, 2009;Newpher et al, 2005;Stimpson et al, 2009;Toshima et al, 2006). Next Sla2, a talin-like domain protein related to mammalian Hip1 and Hip1R, is recruited, followed rapidly by late coat factors Pan1, End3 (two other EH domain proteins) and Sla1 (a SH3 domain protein) Kaksonen et al, 2005;Newpher and Lemmon, 2006).…”

Endocytic adaptors – social networking at the plasma membrane

“…The Syp1 mHD also binds directly to and traffics the transmembrane cargo protein Mid2, which makes Syp1 one the first BAR-domain-containing proteins known to bind cargo. The unstructured middle linker region of Syp1 was also shown to be important in the regulation of actin polymerization through inhibition of Las17 (the yeast ortholog of mammalian WASP) (Boettner et al, 2009).…”