Abstract. The folding of actin and tubulin is mediated via interaction with a heteromeric toroidal complex (cytoplasmic chaperonin) that hydrolyzes ATP as part of the reaction whereby native proteins are ultimately released. Vertebrate actin-related protein (actin-RPV) (also termed centractin) and ~/-tubulin are two proteins that are distantly related to actin and tubulin, respectively: 3,-tubulin is exclusively located at the centrosome, while actin-RPV is conspicuously abundant at the same site. Here we show that actin-RPV and 3,-tubulin are both folded via interaction with the same chaperonin that mediates the folding of/~-actin and o~-and/~-tubulin. In each case, the unfolded polypeptide forms a binary complex with cytoplasmic chaperonin and is released as a soluble, monomeric protein in the presence of Mg-ATP and the presence or absence of Mg-GTE In contrast to a-and/~-tubulin, the folding of 3,-tubulin does not require the presence of cofactors in addition to chaperonin itself. Monomeric actin-RPV produced in in vitro folding reactions cocycles efficiently with native brain actin, while in vitro folded -/-tubulin binds to polymerized microtubules in a manner consistent with interaction with microtubule ends. Both monomeric actin-RPV and ~/-tubulin bind to columns of immobilized nucleotide: monomeric actin-RPV has no marked preference for ATP or GTP, while qr-tubulin shows some preference for GTP binding. We show that actin-RPV and ~/-tubulin compete with one another, and with/~-actin or c~-tubulin, for binary complex formation with cytoplasmic chaperonin.

We describe the structure and expression of a mammalian beta‐tubulin isotype (M beta 6) that is weakly expressed in testis but is abundant in developing brain, with transcripts declining to lower levels in the adult brain. The expression of M beta 6 was undetectable in any other mouse tissue examined. A serum specific for this isotype was prepared using a cloned fusion protein as immunogen. M beta 6 is one of five known beta‐tubulin isotypes expressed in brain, and using the anti‐M beta 6 serum along with sera, anti‐M beta 2, anti‐M beta 3/4 and anti‐M beta 5, previously characterized, we have examined the pattern of expression of beta‐tubulin isotypes in rat cerebellum. The isotypes each have characteristic cell‐type specific patterns of localization in cerebellum. M beta 2, M beta 3/4 and M beta 5 are present in both neuronal and non‐neuronal cells, but in contrast M beta 6 was only detectable in neurons in tissue sections and in dissociated cerebellar cell culture. The majority of sequence differences among the beta‐tubulin isotypes lie at the carboxy terminus, the region of beta‐tubulin involved in MAP binding. In the case of M beta 2 and M beta 6, the patterns of expression are similar or identical to the patterns of expression of MAP3 and MAP1A respectively. These results suggest that beta‐tubulin isotypes may contribute to the determination of the specific association of MAPs with microtubules of diverse function. However, the strict subcellular segregation of other MAPs in brain may be determined by other factors.

The sequence of two human beta-tubulin pseudogenes is described. One contains an intervening sequence but lacks sequences encoding the 55 N-terminal amino acids of the polypeptide chain. A second has no introns but has a polyadenylate signal and an oligoadenylate tract at its 3' end, and it is flanked by a short direct repeat. These sequences have arisen by different mechanisms, including one that probably involves reverse transcription of a processed messenger RNA and reintegration of the complementary DNA copy into the genome.

No abstract