Journal article
Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect
The Journal of cell biology, Vol.123(5), pp.1185-1195
12/01/1993
DOI: 10.1083/jcb.123.5.1185
PMID: 8245125
Abstract
Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.] 347: 44-49) hypothesized that between subdomains 3 and 4 of actin is a loop of 10 amino acids including a four residue hydrophobic plug that inserts into a hydrophobic pocket formed by two adjacent monomers on the opposing strand thereby stabilizing the F-actin helix. To test this hypothesis we created a mutant yeast actin (L266D) by substituting Asp for Leu266 in the plug to disrupt this postulated hydrophobic interaction. Haploid cells expressing only this mutant actin were viable with no obvious altered phenotype at temperatures above 20 degrees C but were moderately cold-sensitive for growth compared with wild-type cells. The critical concentration for polymerization increased 10-fold at 4 degrees C compared with wild-type actin. The length of the nucleation phase of polymerization increased as the temperature decreased. At 4 degrees C nucleation was barely detectable. Addition of phalloidin-stabilized F-actin nuclei and phalloidin restored L266D actin's ability to polymerize at 4 degrees C. This mutation also affects the overall rate of elongation during polymerization. Small effects of the mutation were observed on the exchange rate of ATP from G-actin, the G-actin intrinsic ATPase activity, and the activation of myosin S1 ATPase activity. Circular dichroism measurements showed a 15 degrees C decrease in melting temperature for the mutant actin from 57 degrees C to 42 degrees C. Our results are consistent with the model of Holmes et al. (Holmes, K. C., D. Popp, W. Gebhard, and W. Kabsch. 1990. Nature [Lond.]. 347:44-49) involving the role of the hydrophobic plug in actin filament stabilization.
Details
- Title: Subtitle
- Yeast actin with a mutation in the "hydrophobic plug" between subdomains 3 and 4 (L266D) displays a cold-sensitive polymerization defect
- Creators
- Xin Chen - Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104R Kimberley Cook - Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104Peter A Rubenstein - Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242-1104
- Resource Type
- Journal article
- Publication Details
- The Journal of cell biology, Vol.123(5), pp.1185-1195
- DOI
- 10.1083/jcb.123.5.1185
- PMID
- 8245125
- ISSN
- 0021-9525
- eISSN
- 1540-8140
- Language
- English
- Date published
- 12/01/1993
- Academic Unit
- Stead Family Department of Pediatrics; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984024510302771
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