A mammalian actin substitution in yeast actin (H372R) causes a suppressible mitochondria/vacuole phenotype.
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.
McKane
Melissa
M
Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, Iowa 52242, USA.
Wen
Kuo-Kuang
KK
Boldogh
Istvan R
IR
Ramcharan
Sharmilee
S
Pon
Liza A
LA
Rubenstein
Peter A
PA
D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000495:Allosteric Site; D001120:Arginine / Q000737:chemistry; D002244:Carbon / Q000737:chemistry; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D003599:Cytoskeleton / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009132:Muscles / Q000378:metabolism; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D011108:Polymers / Q000737:chemistry; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011721:Pyrenes / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014617:Vacuoles / Q000378:metabolism*
D000199:Actins; D008301:Maleimides; D011108:Polymers; D011721:Pyrenes; D049452:Green Fluorescent Proteins; C043592:maleimide; D006639:Histidine; D002244:Carbon; D001120:Arginine; C030984:pyrene; D005947:Glucose; D003545:Cysteine; D005990:Glycerol