TY - JOUR
T1 - Purification and polymerization properties of two lethal yeast actin mutants
AU - Frieden, Carl
AU - Du, Jinyan
AU - Schriefer, Lawrence
AU - Buzan, Jenny
N1 - Funding Information:
We thank Robert Horton for technical assistance and Drs. Steven Almo and Sergey Vorobiev for providing the yeast actin X-ray coordinates and for helpful discussions. This work was supported by a National Science Grant MB9603807.
PY - 2000/5/10
Y1 - 2000/5/10
N2 - The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth, The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 μM) without a large change in the ability to form nuclei for the nucleation-elongation process. (C) 2000 Academic Press.
AB - The budding yeast Saccharomyces cerevisiae contains a single actin gene and the gene product, actin, is essential for growth. Two mutants of yeast actin that do not support yeast growth were prepared from yeast by coexpressing the mutant and a 6-histidine-tagged wild-type actin followed by separation of the wild-type and mutant actin using Ni-NTA chromatography as described elsewhere. The mutations, in muscle actin numbering, were at positions 334 (Glu334Lys) and 168 (Gly168Arg) and were chosen based on phenotypic changes observed in the behavior of actin mutants of Caenorhabditis elegans. Glu334 is located on the surface of actin between subdomains 1 and 3 while Gly168 is located in a region near actin-actin contacts in the actin filament. The Glu334Lys mutant polymerized slightly faster than wild-type yeast actin, suggesting that loss of interactions with some actin binding protein, rather than loss of actin-actin contacts, was responsible for its inability to support yeast growth, The Gly168Arg mutant polymerized at a rate similar to wild-type but the extent was considerably less, kinetic characteristics suggesting a high critical concentration (ca. 4 μM) without a large change in the ability to form nuclei for the nucleation-elongation process. (C) 2000 Academic Press.
UR - http://www.scopus.com/inward/record.url?scp=0034630737&partnerID=8YFLogxK
U2 - 10.1006/bbrc.2000.2650
DO - 10.1006/bbrc.2000.2650
M3 - Article
C2 - 10799320
AN - SCOPUS:0034630737
SN - 0006-291X
VL - 271
SP - 464
EP - 468
JO - Biochemical and Biophysical Research Communications
JF - Biochemical and Biophysical Research Communications
IS - 2
ER -