Abstract
Residues 262-274 form a loop between subdomains 3 and 4 of actin. This loop may play an important role in actin filament formation and stabilization. To assess directly the behavior of this loop, we mutated Ser265 of yeast actin to cysteine (S265C) and created another mutant (S265C/C374A) by changing Cys374 of S265C actin to alanine. These changes allowed us to attach a pyrene maleimide stoichiometrically to either Cys374 or Cys265. These mutations had no detectable effects on the protease susceptibility, intrinsic ATPase activity, and thermal stability of labeled or unlabeled G-actin. The presence of the loop cysteine, either labeled or unlabeled, did not affect the actin-activated S1 ATPase activity or the in vitro motility of the actin. Both mutant actins, either labeled or unlabeled, nucleated filament formation considerably faster than wild-type (WT) actin, although the critical concentration was not affected. Whereas the fluorescence of the C-terminal (WT) probe increased during polymerization, that of the loop (S265C/C374A) probe decreased, and the fluorescence of the doubly labeled actin (S265C) was approximately 50% less than the sum of the fluorescence of the individual fluorophores. Quenching was also observed in copolymers of labeled WT and S265C/C374A actins. An excimer peak was present in the emission spectrum of labeled S265C F-actin and in the labeled S265C/C374A-WT actin copolymers. These results show that in the filaments, the C-terminal pyrene of a substantial fraction of monomers directly interacts with the loop pyrene of neighboring monomers, bringing the two cysteine sulfurs to within 18 A of one another. Finally, when bound to labeled S265C/C374A F-actin, myosin S1, but not tropomyosin, caused an increase in fluorescence of the loop probe. Both proteins had no effect on excimer fluorescence. These results help establish the orientation of monomers in F-actin and show that the binding of S1 to actin subdomains 1 and 2 affects the environment of the loop between subdomains 3 and 4.
Highlights
Residues 262–274 form a loop between subdomains 3 and 4 of actin
No significant differences were found in the generation times of the Ser265 of yeast actin to cysteine (S265C), S265C/C374A, and wild-type cells at the temperatures tested
The growth of mutant and WT cells was identical at 30 °C in medium containing 2% ethylene glycol as a sole carbon source, indicating that the inheritance of mitochondria in both S265C and S265C/C374A cells is normal
Summary
Materials—The site-directed mutagenesis kit and [g-32P]ATP (6000 Ci/mmol) were purchased from Amersham Corp. PRS-SC was used to generate diploid yeast cells carrying both the wild-type and mutant actin genes as well as haploid cells containing the mutant sequence as the only actin coding sequence in the cell [24]. The resulting plasmid, pRS-SCCA, was used to generate haploid cells containing the mutant sequence as the only actin coding sequence. Fluorescence Spectroscopy of Pyrene-labeled Wild-type and Mutant Actins—The change in pyrene fluorescence following actin polymerization was observed using either an SLM Model 4800 fluorometer or a Spex fluorolog set at an excitation wavelength of 365 nm, an emission wavelength of 386 nm, and a slit width of 1 mm. Binding of S1 to Actin—The binding of S1 to pyrene-labeled WT and S265C actins was measured at 25 °C in a buffer containing 50 mM KCl, 2 mM MgCl2, and 5.0 mM Tris-HCl, pH 7.8. The binding was monitored as described by Geeves and Jeffries [37] via quenching of pyrene fluorescence of the label attached to Cys374 in both WT and S265C actins
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