Purification and biochemical characterization of tubulin from the budding yeast Saccharomyces cerevisiae.
We describe a method for isolating milligram quantities of assembly-competent tubulin from the budding yeast Saccharomyces cerevisiae. The tubulin is > 95% purified and free of contaminating enzyme activities. As a result, it has been possible to determine the yeast tubulin equilibrium-binding constant for Mg-GTP and the tubulin GTPase activity under nonassembling and assembling conditions. We also determined the critical concentration for yeast tubulin polymerization and found it to be significantly lower than that for bovine brain tubulin under identical conditions. Similarly, the dynamic properties both of individual yeast microtubules and of bulk microtubule suspensions were significantly different from those of bovine brain microtubules free of microtubule-associated proteins. The data suggest that the properties of the yeast tubulin may reflect the particular functional requirements of the yeast cell. With this method, it is now possible to introduce any desired tubulin gene mutation into the budding yeast and correlate the phenotypic effects of the mutation in cells with the effects of the mutation on the biochemical and polymerization properties of the tubulin.
Davis
A
A
Department of Biological Sciences, University of California, Santa Barbara 93106.
Sage
C R
CR
Wilson
L
L
Farrell
K W
KW
D001665:Binding Sites; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D011108:Polymers; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011108:Polymers; D014404:Tubulin; C036852:magnesium GTP; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases