Beryllium Fluoride and Phalloidin Restore Polymerizability of a Mutant Yeast Actin (V266G,L267G) with Severely Decreased Hydrophobicity in a Subdomain 3/4 Loop*
- ‡ To whom correspondence should be addressed: Dept. of Biochemistry, College of Medicine, 4403 Bowen Science Bldg., Iowa City, IA 52242-1109. Tel.: 319-335-7911; Fax: 319-335-9570.
Abstract
Holmes proposed that in F-actin, hydrophobic residues in a subdomain 3/4 loop interact with a hydrophobic pocket on the opposing strand resulting in helix stabilization. We have determined how a decreased hydrophobicity of this plug affects yeast actin function. Cells harboring only the V266G, V266D, V266F, L267G, L269D, or L269K actins appear normal, although V266G cells display an altered budding pattern. However, V266G,L267G (GG) double mutant cells are cold-sensitive with randomly oriented thick actin assemblies seen in rhodamine phalloidin-stained GG cells. V266D actin polymerizes slower than wild-type actin at room temperature. At 4°C, not only is polymerization slowed, but there is also an effect on critical concentration. However, the polymerization defects are milder than those associated with substitution of Asp for the neighboring Leu267. Purified GG-actin does not polymerize in vitro alone or in the presence of wild-type F-actin seeds. GG-actin polymerization can be restored by larger amounts of wild-type actin, beryllium fluoride, or phalloidin at room temperature, although at 4°C only phalloidin is effective. These results suggest that the diminished hydrophobicity of the plug in GG-actin leads to filament destabilization. However, the V266D actin results require a modification of the original Holmes filament model.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM33689 (to P. A. R.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1The L267D mutation was originally noted as L266D (17) to reflect the fact that we called the initiator Met residue of actin Met-1. However, since the initiator Met is retained in yeast actin, we have renumbered this residue.
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↵2 The abbreviations used are:
- Cc
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critical concentration
- DAPI
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4′,6′-diamidino-2-phenylindole
- FM 4-64
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N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide
- BeFx
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beryllium fluoride
- WT
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wild type.
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↵3K. Hill and L. Weisman, personal communication.
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↵4Kuang, B., and Rubenstein, P. A. (1997) J. Biol. Chem. 272, in press.
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- Received July 9, 1996.
- Revision received October 14, 1996.
- © 1997 by The American Society for Biochemistry and Molecular Biology, Inc.
