In vivo importance of actin nucleotide exchange catalyzed by profilin - PubMed (original) (raw)
In vivo importance of actin nucleotide exchange catalyzed by profilin
A K Wolven et al. J Cell Biol. 2000.
Abstract
The actin monomer-binding protein, profilin, influences the dynamics of actin filaments in vitro by suppressing nucleation, enhancing nucleotide exchange on actin, and promoting barbed-end assembly. Profilin may also link signaling pathways to actin cytoskeleton organization by binding to the phosphoinositide PIP(2) and to polyproline stretches on several proteins. Although activities of profilin have been studied extensively in vitro, the significance of each of these activities in vivo needs to be tested. To study profilin function, we extensively mutagenized the Saccharomyces cerevisiae profilin gene (PFY1) and examined the consequences of specific point mutations on growth and actin organization. The actin-binding region of profilin was shown to be critical in vivo. act1-157, an actin mutant with an increased intrinsic rate of nucleotide exchange, suppressed defects in actin organization, cell growth, and fluid-phase endocytosis of pfy1-4, a profilin mutant defective in actin binding. In reactions containing actin, profilin, and cofilin, profilin was required for fast rates of actin filament turnover. However, Act1-157p circumvented the requirement for profilin. Based on the results of these studies, we conclude that in living cells profilin promotes rapid actin dynamics by regenerating ATP actin from ADP actin-cofilin generated during filament disassembly.
Figures
Figure 1
Space filling model of yeast profilin, showing three rotations. Residues mutated in this study are indicated by arrows. Residues are color coded to illustrate function. Residues predicted to contact actin are red and orange. Orange residues were mutated to alanine. Residues forming the polyproline binding site are blue and green. Blue residues are hydrophobic and are predicted to have stronger effects on polyproline binding than green residues. Conserved basic residues and acidic residues are colored yellow and purple, respectively.
Figure 2
Rhodamine-phalloidin staining of yeast expressing pfy1-4 and act1-157. Yeast cells were grown to log phase in rich media at 25°C then fixed with formaldehyde, or were shifted to 37°C for 3 h, and then fixed. The F-actin was stained with rhodamine phalloidin. Yeast expressing PFY1 (A), pfy1-4 (B), pfy1-4 and act1-157 (C), and act1-157 (D). Bar, 10 μm.
Figure 3
Uptake of Lucifer yellow by fluid-phase endocytosis in yeast expressing pfy1-4 and act1-157. Yeast cells were grown to early log phase at 25°C and some were shifted to 37°C. After 2 h, uptake of Lucifer yellow into the vacuole was monitored by fluorescence microscopy. Yeast expressing PFY1 (A), pfy1-4 (B), pfy1-4 and act1-157 (C), and act1-157 (D). Note that in the periphery of B at 37°C, very bright Lucifer yellow staining of dead cells expressing pfy1-4 is evident. Bar, 10 μm.
Figure 4
Profilin binding to monomeric actin. Purified yeast actin and recombinant yeast profilin were incubated together to examine actin monomer binding. A, PLP–Sepharose cosedimentation assay. Various concentrations of Pfy1p and Pfy1-4p were bound to PLP–Sepharose and then incubated with 5 μM actin. Actin bound to the beads (B) was separated from the free actin (F) by centrifugation and analyzed by SDS-PAGE and Coomassie staining. B, Quantitation of results from the PLP–Sepharose cosedimentation experiment using various combinations of Act1p, Act1-157p, Pfy1p, and Pfy1-4p. Shown is a plot of the percent of actin bound versus profilin concentration. C, Actin assembly assay. Increasing concentrations of Pfy1p or Pfy1-4p were incubated with 5 μM Act1p or Act1-157p. Steady state reactions were centrifuged, analyzed by SDS-PAGE, and quantitated. D, Urea denaturation of Pfy1p and Pfy1-4p.
Figure 5
Actin filament turnover assays and steady state assembly. In A and B, purified Act1p, Act1-157p, Pfy1p, Pfy1-4p, and Cof1p were incubated in various combinations at a concentration of 25 μM for each protein. Actin assembly was initiated by the addition of polymerization salts, and phosphate released from actin filaments was measured. Rates of actin treadmilling are measured by comparing the slopes of the linear portions of the graph for each reaction, after steady state levels of assembly have been reached. The numbers in parentheses represent average rates of treadmilling from several experiments, expressed in pmol phosphate/s. A, Actin treadmilling using Act1p. B, Actin treadmilling using Act1-157p. C, Actin assembly in the presence of Pfy1p and Cof1p. Reactions were performed as in Fig. 4 C, except Cof1p was included in each reaction at a concentration of 5 μM.
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