Overexpression of tubulin in yeast: differences in subunit association.
Microtubules are cytoskeletal organelles composed principally of polymerized alpha beta-tubulin heterodimers. The specific roles and the detailed structures of the individual alpha- and beta-tubulin subunits have not been established, since the conditions necessary for separating the heterodimer result in loss of the subunits' ability to repolymerize. We have overcome this obstacle by constructing plasmids which allow regulated overexpression of individual tubulin subunits in the yeast Saccharomyces cerevisiae under control of the galactose promoter. Overproduction was monitored with alpha- and beta-tubulin-specific antibodies using immunoblotting of cell extracts, and the state of association of the individual subunits in vivo was determined by immunofluorescence microscopy. Cells overproducing only beta-tubulin accumulated fibrous structures associated with the cell membrane, whereas cells overproducing only alpha-tubulin displayed a diffuse signal throughout the cytoplasm. Cells simultaneously overexpressing alpha- and beta-tubulin subunits accumulated membrane-associated, filamentous arrays in which both subunits were incorporated. When cells with the fibrous tubulin-containing structures are treated with zymolyase, a yeast cell wall disrupting enzyme, the fibers appear to splay apart, suggesting that the immunofluorescent rings represent bundles of fibers. Cells overproducing beta-tubulin alone or both alpha- and beta-tubulin were examined at various times after galactose induction, and significant differences were found in the tubulin association state prior to the formation of fibers. For alpha beta-tubulin, fibers form directly from a nuclear structure, whereas beta-tubulin alone first accumulates in the cytoplasm. The differences in patterns of tubulin accumulation and assembly presumably reflect a difference in the intrinsic association properties of the alpha- and beta-subunits.
Department of Biochemistry, University of Geneva, Switzerland.
D M
DM
Bollag
I
I
Tornare
R
R
Stalder
A M
AM
Paunier Doret
M D
MD
Rozycki
S J
SJ
Edelstein
D000906:Antibodies; D005455:Fluorescent Antibody Technique; D015966:Gene Expression Regulation, Fungal; D046911:Macromolecular Substances; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014404:Tubulin / Q000096:biosynthesis / Q000378:metabolism*
D000906:Antibodies; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014404:Tubulin