Mutants of chinese hamster ovary cells affected in two different microtubule-associated proteins. Genetic and biochemical studies

Abstract

Several colchicine-resistant (ColR) mutants of a Chinese hamster ovary cell line, PodRII6, which has earlier been shown to be affected in a microtubule-associated protein (referred to as P1 in these studies), have been isolated. Gel electrophoretic analyses show that one of the ColR mutants (ColR22) contains an additional new protein spot (designated as M2; Mr congruent to 70,000) that is derived by a mutational alteration in a neighboring more acidic protein P2 present in both PodRII6 and ColR22 cell lines. Studies on the relative amounts of the M2 and P2 proteins (and also the M1 and P1) in the ColR22 mutant and hybrid (i.e. ColR22 X PodS) cells are in accordance with the codominant behavior of the mutations and provide evidence that in the ColR22 mutant only one of the two copies of the genes for these proteins has been mutated. The two proteins which are affected in this mutant have been shown to constitute microtubule-associated proteins by a number of criteria. (i) These proteins along with tubulins are specifically released upon Ca2+ treatment of detergent-extracted cytoskeletons containing microtubules, but not if microtubules have been destroyed by prior treatment with colchicine. (ii) Cold treatment of a microtubule-containing fraction also releases the same set of proteins as in criterion i above. (iii) When the mutant cells are treated as in criterion i or ii, then along with the wild type forms, the mutant forms of the above proteins are also found in the microtubule-specific fractions. The cell extracts from PodRII6 and ColR22 mutants show reduced binding of both [3H] podophyllotoxin and [3H]colchicine, indicating that the above proteins are involved in the binding of these drugs to microtubules. It is suggested that the proteins P1 and P2, which are present in Chinese hamster ovary cells in nearly equimolar amounts with (alpha + beta)-tubulins, may comprise important microtubule structural components.