Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis.
gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.
Hendrickson
T W
TW
Program in Biochemistry, Cell, and Developmental Biology, Department of Cell Biology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Yao
J
J
Bhadury
S
S
Corbett
A H
AH
Joshi
H C
HC
D000595:Amino Acid Sequence; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division / Q000502:physiology*; D020090:Chromosome Segregation / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000235:genetics / Q000502:physiology*; D008941:Spindle Apparatus / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D014404:Tubulin