Structure and Mechanism of Mouse Cyclase-associated Protein (CAP1) in Regulating Actin Dynamics

Abstract

Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding β-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.

Srv2/CAP is required for polarized actin cable assembly and patch internalization during clathrin-mediated endocytosis

“…7A-C, lower panels and graph; Movie 7). This observation is consistent with the reported interactions of the HFD domain with F-actin (Jansen et al, 2014). These results suggest that localization of Srv2p to cortical patches through the HFD domain might depend on the nucleotide-bound state of actin, or a protein that has a preferential affinity for ADP-actin.…”

“…First, deletion of the PWP region, even in abp1Δ cells, did not completely impair the cortical localization of Srv2p, but deletion of the HFD domain of Srv2p further decreased the cortical localization of Srv2p when expressed in abp1Δ cells. As the HFD domain binds directly to F-actin, in addition to its actindependent interaction with cofilin (Jansen et al, 2014), the HFD interaction with F-actin also probably contributes to Srv2p localization. Second, Srv2p and Abp1p exhibited apparently different localizations in sla2Δ cells, with Abp1p stably associating with the actin comet tail and actively treadmilling through the elongated actin structures (Kaksonen et al, 2003;Okreglak and Drubin, 2007), whereas Srv2p localized only at the cytosolic edges of the actin tail.…”

“…These functions are accomplished through a high-affinity association of the Srv2p C-terminus with ADP-G-actin, which releases the actin from cofilin (Balcer et al, 2003;Balderhaar et al, 2013;Chaudhry et al, 2010;Mattila et al, 2004;Moriyama and Yahara, 2002). By contrast, the N-terminal oligomerization and the HFD domains can catalyze cofilin-mediated severing of actin filaments (Chaudhry et al, 2013;Jansen et al, 2014). In addition, several proteins have been reported to selectively bind to either the P1 or P2 regions of Srv2p; profilin, a nucleotide exchange factor for actin, binds to P1 (Bertling et al, 2007), and Abp1p, an actin-binding protein, binds to P2 (Freeman et al, 1996;Lila and Drubin, 1997).…”

CASEIN KINASE1-LIKE PROTEIN2 Regulates Actin Filament Stability and Stomatal Closure via Phosphorylation of Actin Depolymerizing Factor

“…The flow chamber was prepared as described previously (Amann and Pollard, 2001;Jansen et al, 2014). For actin-filament-severing assays, the assembled flow chamber was incubated with 25 nM N-ethylmaleimide-myosin for 5 min followed by washing with 1% BSA for 3 min.…”

“…Single actin filament severing was directly visualized by TIRF microscopy as described previously (Amann and Pollard, 2001;Andrianantoandro and Pollard, 2006;Jansen et al, 2014). The flow chamber was prepared as described previously (Amann and Pollard, 2001;Jansen et al, 2014).…”