Abstract
With yeast actin, contrary to other actins, filament formation, ATP hydrolysis, and Pi release are concurrent at low actin concentrations, the condition usually employed to assess actin polymerization. This observation leads to a question concerning the conformation of the filament barbed end that might be recognized by specific actin-binding proteins. To try to detect possible new actin polymer conformations that might be intermediate in the pathway leading to mature F-actin, we monitored the change in intrinsic tryptophan fluorescence of yeast and muscle actins polymerized at pH 6 to accelerate the rate of filament formation. This allowed temporal resolution of the Pi release process from the slower process of polymerization. With both actins, we detected a biphasic instead of the usual monophasic fluorescence change, a rapid decrease that tracks with filament formation followed by a slower rebound (the second phase). This second phase postpolymerization conformational change requires Pi release and occurs nearly coincident with its release. The addition of Pi causes this second phase response to disappear, and the inclusion of Pi during polymerization prevents its appearance. At pH 7.5, with higher yeast actin concentrations to accelerate polymerization, a two-phase fluorescence change is also observed. In this case, the second phase change lags substantially behind Pi release. Pi release could also be resolved from polymer formation. V159N yeast actin, hypothesized previously as remaining in a postpolymerization ATP-like state, exhibits the same two-phase intrinsic tryptophan fluorescence behavior as wild-type yeast actin. Together, these observations demonstrate the presence of an intermediate filament state between ADP-Pi and mature ADP-F-actin.
Highlights
Actin binds a molecule of adenine nucleotide with high affinity in the central cleft separating the two domains of the protein [2]
Intrinsic Tryptophan Fluorescence Behavior of Polymerizing Yeast Actin—To accelerate actin polymerization relative to Pi release to determine whether we could temporally resolve the two processes, we carried out polymerization of yeast actin at pH 6.0 based on the previous work of Zimmerle and Frieden [34]
Doyle et al [38], using site-directed mutagenesis of yeast actin, demonstrated that the polymerization-dependent fluorescence decrease is the combined contribution of three of the four tryptophan residues, Trp-79, Trp-86, and Trp-340, all of which are near the nucleotide binding site in subdomain 1
Summary
Actin binds a molecule of adenine nucleotide with high affinity in the central cleft separating the two domains of the protein [2]. The recent work of Yao and Rubenstein [27] indicates that with yeast actin neither the ATP- nor the ADP-Pi state exists for any appreciable amount of time in actively growing filaments when polymerization is carried out at low actin concentrations.
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