POLYMERIZATION AND IN-VITRO MOTILITY PROPERTIES OF YEAST ACTIN - A COMPARISON WITH RABBIT SKELETAL ALPHA-ACTIN

Actin purified from the yeast (Saccharomyces cerevisae) was polymerize d faster than rabbit skeletal alpha-actin by MgCl2. The two actins pol ymerized at similar rates in the presence of CaCl2. Yeast actin, up to 25 mu M, was not polymerized by KCl (100-300 mM); the monovalent salt also inhibited the MgCl2-induced polymerization of actin. The local s tructure of the subdomain-2 region in yeast actin filaments was probed by subtilisin and trypsin digestions. Loop 38-52 appeared more flexib le and accessible to subtilisin in yeast than in rabbit actin. In cont rast, tryptic digestions at Lys-61 and -68 occurred at the same rate f or yeast and alpha-actin filaments. Modification of yeast actin by a s ulfhydryl reagent CPM thylamino)-3-(4'-maleimidophenyl)-4-methylcoumai n] was specific to the Cys-374 residue; no labeling of a yeast actin m utant containing an alanine substitution for cysteine 374 was observed , The rates of Cps-374 labeling by CPM were similar for yeast and musc le actin, suggesting a similar environment for the C terminus in both polymers. In the in vitro motility assays, yeast actin required higher concentrations of heavy meromyosin (HMM) for its sliding than did the rabbit actin. At saturating concentrations of HMM, the sliding veloci ties of both actins were the same (3.0 mu m/s), Relative forces genera ted by HMM with yeast and muscle actin were assessed by monitoring the ir in vitro motility in the presence of NEM-HMM load. The sliding of y east actin was stopped at a level of external load (molar ratio NEM-HM M/HMM = 0.25) lower than that of muscle actin (NEM-HMM/HMM = 0.43), su ggesting lower force production with yeast actin. These results are di scussed in terms of the myosin cross-bridge cycle and actomyosin inter actions.