Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Abstract

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective ,-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a 0-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered j3-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as P-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective I8-tubulins of lower apparent molecular weight. The altered mobility of these I-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35SImethionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for 0-tubulin. Assembly-defective ,-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both a-and 0-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a ,-tubulin gene product that is not essential for survival.Microtubule assembly is a complex process in which oaand 3-tubulin heterodimers interact end to end and laterally to form hollow tubes 25 nm in diameter. Much of our understanding of microtubule assembly has come from in vitro experiments pioneered by Weisenberg (29). These and subsequent studies have demonstrated a requirement for GTP, a pH optimum of 6.6 to 6.9, and a minimum concentration of 0.2 to 1 mg of tubulin per ml. In addition, enhanced polymerization occurs in the presence of glycerol, dimethyl sulfoxide, polyethylene glycol, D20, polycations, and microtubule-associated proteins; but polymerization is inhibited by Ca2+ (10). More recent studies using proteolytic cleavage of tubulin by subtilisin have shown that a highly acidic C-terminal 4-kilodalton (kDa) fragment can be released from both oa-and ,B-tubulin, resulting in a tubulin species which assembles at a lower critical concentration, fails to bind microtubule-associated proteins, and is insensitive to Ca2+ These in vitro studies have greatly expanded our knowledge of factors that affect tubulin assembly and have provided valuable information on the function of particular subunit domains on tubulin. Comparable in vivo techniques for approaching similar questions have not been well developed. In particular, a genetic approach toward studying assembly has not yet been used, even though such an approach has proved to be invaluable for under...

Altered Expression of Mβ2, the Class II β-Tubulin Isotype, in a Murine J774.2 Cell Line with a High Level of Taxol Resistance

“…The latter method also detected an increased expression of ␣-tubulin that may reflect coordinate regulation of the intracellular levels of ␣-and ␤-tubulin as has been suggested previously (52). Alternatively, it may be that either qualitative or quantitative alterations in ␣-tubulin can in their own right contribute to the taxol-resistant phenotype.…”

A Ubiquitous β-tubulin Disrupts Microtubule Assembly and Inhibits Cell Proliferation

“…In this process, many genes were lost or became haploid (Siminovitch, 1976). In addition, we have shown that CHO cells produce a surfeit of tubulin and can lose half of their ␤1-tubulin (35% of total ␤-tubulin) with no apparent effect on growth of the cells (Boggs and Cabral, 1987). Yet, the low percentage of ␤5-tubulin has persisted, suggesting that this isotype may play a vital role in microtubule assembly.…”

Interaction of the RhoA Exchange Factor Net1 with Discs Large Homolog 1 Protects It from Proteasome-mediated Degradation and Potentiates Net1 Activity

“…3 Protein translation was then inhibited for different periods of time using cyclohexamide, following which the cells were lysed, and levels of the transfected proteins were determined by Western blotting. As a control, we also measured the levels of ␣-tubulin, which has a half-life of greater than 40 h (25). In these assays, we observed that Net1A expressed together with GFP exhibited a half-life of 0.35 h (Fig.…”