Expression of amino- and carboxyl-terminal gamma- and alpha-tubulin mutants in cultured epithelial cells.
Three distinct tubulin proteins are essential for microtubule function: alpha-, beta-, and gamma-tubulin. After translation, alpha- and beta-tubulin proteins combine into a soluble, 7 S heterodimer that is multimerized to form the microtubule filament. Conversely, gamma-tubulin combines with several proteins into a soluble, 25 S multi-protein particle, the gammasome that is essential for nucleating microtubule filaments at the centrosome. The proteins that assist tubulins in executing their specific functions are largely unknown. As an initial approach to address this issue, we first decided to identify domains of mammalian alpha- and gamma-tubulin necessary for their function by creating mutant mammalian alpha- and gamma-tubulin (both deletion and hybrid mutants) and assaying their behavior in stably transfected Chinese hamster ovary epithelial cells. First, we demonstrated that addition of a carboxyl-terminal epitope tag had no effect on the subcellular localization of either alpha- and gamma-tubulin. Second, we found that both the amino and carboxyl termini of gamma-tubulin were essential for its incorporation into the gammasome. Third, we found that the amino and carboxyl termini of alpha-tubulin were necessary for incorporation of the alpha-beta-tubulin heterodimer into the microtubule filament network. In general, alpha-tubulin sequences could not replace those of gamma-tubulin and vice versa. Taken together, these results suggest that the amino and carboxyl termini of alpha- and gamma-tubulin and perhaps regions throughout these proteins were necessary for their specific functions.
Leask
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FibroGen, Inc., South San Francisco, California 94080-6902, USA. aleask@fibrogen.com
Stearns
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T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D018385:Centrosome / Q000737:chemistry; D006224:Cricetinae; D019281:Dimerization; D004847:Epithelial Cells; D006801:Humans; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D015335:Molecular Probes; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013045:Species Specificity; D013329:Structure-Activity Relationship; D014162:Transfection; D014404:Tubulin / Q000235:genetics / Q000378:metabolism*
D015335:Molecular Probes; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D011993:Recombinant Fusion Proteins; D014404:Tubulin