A central role for the WH2 domain of Srv2/CAP in recharging actin monomers to drive actin turnover in vitro and in vivo

Abstract

Cellular processes propelled by actin polymerization require rapid disassembly of filaments, and then efficient recycling of ADF/cofilin-bound ADP-actin monomers back to an assembly-competent ATP-bound state. How monomer recharging is regulated in vivo is still not well understood, but recent work suggests the involvement of the ubiquitous actin-monomer binding protein Srv2/CAP. To better understand Srv2/CAP mechanism, we explored the contribution of its WH2 domain, the function of which has remained highly elusive. We found that the WH2 domain binds to actin monomers and, unlike most other WH2 domains, exhibits similar binding affinity for ATP-actin and ADP-actin (K(d) approximately 1.5 microM). Mutations in the WH2 domain that impair actin binding disrupt the ability of purified full-length Srv2/CAP to catalyze nucleotide exchange on ADF/cofilin-bound actin monomers and accelerate actin turnover in vitro. The same mutations impair Srv2/CAP function in vivo in regulating actin organization, cell growth, and cell morphogenesis. Thus, normal cell growth and organization depend on the ability of Srv2/CAP to recharge actin monomers, and the WH2 domain plays a central role in this process. Our data also reveal that while most isolated WH2 domains inhibit nucleotide exchange on actin, WH2 domains in the context of intact proteins can help promote nucleotide exchange.

Figure 1. Mutational analysis of the WH2 domain of Srv2/CAP

(A) Schematic of Srv2/CAP domain organization and fragments purified for biochemical analysis in this study. Relevant binding partners are shown above each domain. (B) Alignment of P1-WH2-P2 domain sequences from diverse Srv2/CAP homologues using ClustalW. M. mus1, mouse CAP1; M. mus2, mouse CAP2; D. mel., Drosophila melanogaster CAP; A. tha, Arabidopsis thaliana CAP; and S. cer., S. cerevisiae Srv2. Specific residues changed to alanine are marked A for each allele (srv2-96 through srv2-99). A black bar denotes the WH2 domain of S. cerevisiae Srv2 (residues 295-349). Blue bars denote the two proline-rich regions (P1 and P2). (C) Immunoblot of whole cell extracts from wild type SRV2 and srv2 mutant strains probed with anti-Srv2 antibodies. D) SRV2 and srv2 mutant strains were grown to log phase, serially diluted, plated on YPD plates, and grown at 25°C and 37°C.

Figure 2. Cell morphology defects and genetic interactions of srv2 mutants

(A) DIC imaging of wild type SRV2 and srv2 mutant cells. Cells were grown to mid-log phase at 25°C or 37°C and fixed. (B) Mother cell length/width ratio of wild type SRV2, srv2Δ, and mutant srv2 cells grown at 25°C were determined using CalMorph freeware and averaged (n=100 cells each). (C) Genetic interactions of srv2 alleles with cof1-19. Haploid yeast strains carrying integrated srv2 alleles were crossed to the haploid cof1-19 strain. Diploids were sporulated and tetrads dissected (minimum 20 tetrads, 80 spores), and haploid progeny were compared for cell growth after serial dilution on YPD plates and growth at 25, 30, 34, and 37°C. For each cross, we determined the percentage of haploid progeny compared to a wild type strain that exhibited impaired growth at 37°C (TS), impaired growth at all temperatures (‘Sick’), or were dead at 25°C (‘Dead’).

Figure 3. Cellular actin organization defects in srv2 mutants

Wild type SRV2 and mutant strains were grown in YPD medium to log phase at 25°C and 37°C, fixed, and stained with Alexa488-phalloidin to visualize filamentous actin.

Figure 4. G-actin binding activities of wild type and mutant Srv2 proteins

(A) Coomassie stained gel of full-length wild type and mutant Srv2 proteins. (B) The stability of Srv2 proteins was compared using a fluorescence-monitored urea denaturation assay. The normalized fluorescence is shown on the y-axis and urea concentration on the x-axis. Srv2-WT, Srv2-97, and Srv2-98 unfold at ~5 M urea, while Srv2-99 unfolds at ~6 M urea. (C and D) Monomeric actin (3 μM, 5% pyrene labeled) was polymerized in the presence of different concentrations of wild type and mutant Srv2 proteins (μM indicated by each curve). (E) Rates of inhibition of actin polymerization were plotted against concentration of wild type or mutant Srv2 protein.

Figure 5. G-actin binding affinities of wild type and mutant WH2 peptides

(A) Coomassie stained gel of two WH2 domain peptides (a.a. 295-349 and 306-349). (B) Monomeric actin (3 μM, 5% pyrene labeled) was polymerized in the presence of different concentrations of wild type and mutant WH2 peptides (μM indicated by each curve). (C) Coomassie stained gel of WH2-97 and WH2-98 peptides (a.a. 295-349) (D) Comparison of wild type and mutant WH2 peptide effects on rate of actin polymerization (conditions as in B).

Figure 6. WH2 peptide binding affinities for ATP- and ADP-G-actin

Dissociation constants (K_d shown in each graph) were determined by averaging data from multiple experiments (n=3 each), in which we determined the concentration-dependent effects of WH2 peptides (A and B) or Cof1 (C and D) on rate of nucleotide exchange on ADP-G-actin or ATP-G-actin. Data were fit with a Quadratic Function (see Methods) to derive K_d values.

Figure 7. Role of the WH2 domain in promoting nucleotide exchange on ADF/cofilin-bound ADP-actin monomers

All assays contained 2 μM ADP-G-actin and 5 μM Cof1, except where noted. (A) Comparison of the effects of full-length wild type Srv2 protein (Srv2-WT) and profilin on rate of nucleotide exchange. (B) Comparison of the effects of Srv2-WT and mutant Srv2-97 and Srv2-98 proteins on rate of nucleotide exchange. (C) Comparison of the effects of different fragments of Srv2 (400 nM) on rate of nucleotide exchange. (D) Comparison of the abilities of wild type and mutant WH2 peptides to restore nucleotide exchange activity to full-length Srv2-98 protein. (E) Effects of different domains of Srv2, alone and in combination (400 nM or 4 μM, as indicated), on the nucleotide exchange rate of Cof1-bound ADP-G-actin. (F) Effects of yeast profilin (5 μM) on the nucleotide exchange rate of ADP-G-actin in the presence and absence of full-length Srv2-WT (400 nM) and/or Cof1 (5 μM).

Figure 8. Role of the WH2 domain in promoting actin turnover in the presence of ADF/cofilin

All assays contained 8 μM F-actin and 4 μM Cof1. (A) Comparison of full-length wild type and mutant Srv2 proteins (0-500 nM) effects on rate of steady state actin turnover measured by rate of P_i release. (B) Comparison of effects of different fragments of Srv2 (0-500 nM) on rate of actin turnover.