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A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype
Journal article   Open access   Peer reviewed

A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype

Melissa McKane, Kuo-Kuang Wen, Istvan R Boldogh, Sharmilee Ramcharan, Liza A Pon and Peter A Rubenstein
The Journal of biological chemistry, Vol.280(43), pp.36494-36501
10/28/2005
DOI: 10.1074/jbc.M506970200
PMID: 16118223
url
https://doi.org/10.1074/jbc.M506970200View
Published (Version of record) Open Access

Abstract

To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.
 Temperature Actins - metabolism Crystallography, X-Ray Muscle, Skeletal - metabolism Carbon - chemistry Saccharomyces cerevisiae - metabolism Time Factors Actins - chemistry Glycerol - chemistry Muscles - metabolism Protein Structure, Tertiary Green Fluorescent Proteins - metabolism Models, Molecular Mitochondria - metabolism Cysteine - chemistry Arginine - chemistry Phenotype Pyrenes - chemistry Mutagenesis Allosteric Site Cytoskeleton - metabolism Glucose - metabolism Vacuoles - metabolism Polymers - chemistry Protein Conformation Mutation Histidine - chemistry Maleimides - chemistry Microscopy, Fluorescence

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