In vivo discrimination among beta-tubulin isotypes: selective degradation of a type IV beta-tubulin isotype following overexpression in cultured animal cells.
Preceding efforts have revealed that tubulin synthesis in animal cells is regulated both by selective expression of individual members of the tubulin multigene families and by a post-transcriptional control pathway that cotranslationally degrades tubulin mRNAs when the concentrations of unassembled subunits are increased. To test the effect of forced expression of a specific beta-tubulin, we constructed Chinese hamster ovary (CHO) cell lines that stably express the chicken class IV (c-IV) beta-tubulin gene. After gene amplification, we obtained lines that synthesize the c-IV polypeptide at a rate two to three times that of all endogenous beta-tubulins. Despite this elevated rate of synthesis, these c-IV polypeptides accumulated to only 4 to 10% of cellular beta-tubulin. Furthermore, when c-IV transcription was further elevated transiently, there was a compensatory loss in the endogenous class IV isotype (m-IV) so that the total level of class IV isotypes remained unchanged. The data indicate that beta-tubulin isotypes I and IV are biochemically distinguished in these cultured cell lines and that the stability of individual isotypes is established in part by isotype-specific interactions with other cellular factors.
Sisodia
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Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205.
Gay
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DA
Cleveland
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DW
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002645:Chickens / Q000235:genetics; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D008870:Microtubules / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D010053:Ovary; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014404:Tubulin / Q000145:classification / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D014404:Tubulin