Studies of cellular control of microtubule assembly have focused primarily on the assembly reaction from α/β-tubulin heterodimers to microtubule polymers and on the identification of protein cofactors and structures that modulate this polymerization (8
). Results obtained by several approaches suggest that cells may also regulate microtubule morphogenesis at stages preceding the polymerization reaction. Of particular interest are proteins that appear to interact with the α- xand β-tubulin polypeptides and modulate their activities. We are studying these proteins in the yeast Saccharomyces cerevisiae
in order to understand their in vivo functions.
One of these yeast proteins is Rbl2p. Identified in a search for proteins that, when overexpressed, rescue cells from the toxicity of free β-tubulin (5
), Rbl2p binds monomeric β-tubulin to form a heterodimer that excludes α-tubulin, both in vivo and in vitro (5
). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized β-tubulin before it is incorporated into α/β-tubulin heterodimers and β-tubulin released by dissociation of heterodimers (4
). However, the precise function of Rbl2p in vivo is not known.
Biochemical experiments with the vertebrate homolog of Rbl2p, cofactor A, suggest one possible function. Cofactor A was purified from extracts based on its activity in an in vitro tubulin-folding assay that monitors the exchange of tubulin polypeptides released from the cytosolic chaperonin Tri-C into preexisting α/β-tubulin heterodimers (14
). Five cofactors facilitate this reaction. Three of them—cofactors C, D, and E—are necessary for the reaction. The functions of the other two—cofactors A and B—are a subset of the functions of cofactors D and E, respectively, and are not essential in the assay. However, their presence substantially stimulates the reaction (approximately fourfold for cofactor A [21
These experiments also suggest a pathway for the exchange reaction between unfolded tubulin polypeptides and heterodimers. When β-tubulin polypeptides are released from the cytosolic chaperonin, they are able initially to bind either cofactor A or cofactor D but all of the β-tubulin must subsequently be transferred to cofactor D in order to become competent to participate in heterodimer formation. In a parallel pathway, α-tubulin polypeptides released from the cytosolic chaperonin bind to either cofactor B or cofactor E. Those polypeptides that bind cofactor B are then transferred to cofactor E. The cofactor E/α-tubulin complex associates with the cofactor D/β-tubulin complex to generate α/β-tubulin polypeptides that are competent to exchange with exogenous, preexisting heterodimer.
Independently, the S. cerevisiae
genes encoding homologs of four of these cofactors were identified in screens for a wide range of microtubule functions: sensitivity to microtubule-depolymerizing drugs (28
), chromosome instability (18
), sensitivity to undimerized β-tubulin (5
), and functions of mitotic motors (15
). Sequence homology identified the remaining cofactor homolog (11
). The mutant phenotypes produced by deletion of these genes argue against a role for them in the primary pathway for tubulin heterodimer formation, as suggested by the in vitro results, because S. cerevisiae
cells from which the cofactor homolog genes have been deleted, either singly or in combinations, are all viable (5
). Therefore, these cofactors are not required for the formation of tubulin heterodimers in the cell. There may be other genes, as yet undiscovered, that fulfill this function or are redundant with respect to the genes encoding the cofactor homologs. It is also possible that the catalysis of tubulin folding is not required in vivo. In this paper, we address specifically the in vivo role of Rbl2p/cofactor A. Rbl2p expression becomes essential in cells containing an excess of β-tubulin relative to α-tubulin. The data demonstrate that this essential activity of Rbl2p/cofactorA is not displayed by and does not depend upon Cin1p/cofactor D. Therefore, Rbl2p does not rescue cells from β-tubulin lethality by the pathway proposed for the in vitro exchange reaction described above. The results also demonstrate that increasing but substoichiometric levels of Rbl2p are necessary to rescue cells from increasing levels of free β-tubulin. Although Rbl2p is required for this activity, almost all of the cells’ free β-tubulin incorporates into an aggregate that is itself neither detrimental nor sufficient to suppress the toxicity of free β-tubulin. The results suggest that Rbl2p/cofactor A functions to protect cells from free β-tubulin by binding transiently to a subset of the free β-tubulin until it associates with an aggregate.