Formation and function of the Rbl2p-beta-tubulin complex

Abstract

The yeast protein Rbl2p suppresses the deleterious effects of excess beta-tubulin as efficiently as does alpha-tubulin. Both in vivo and in vitro, Rbl2p forms a complex with beta-tubulin that does not contain alpha-tubulin, thus defining a second pool of beta-tubulin in the cell. Formation of the complex depends upon the conformation of beta-tubulin. Newly synthesized beta-tubulin can bind to Rbl2p before it binds to alpha-tubulin. Rbl2p can also bind beta-tubulin from the alpha/beta-tubulin heterodimer, apparently by competing with alpha-tubulin. The Rbl2p-beta-tubulin complex has a half-life of approximately 2.5 h and is less stable than the alpha/beta-tubulin heterodimer. The results of our experiments explain both how excess Rbl2p can rescue cells overexpressing beta-tubulin and how it can be deleterious in a wild-type background. They also suggest that the Rbl2p-beta-tubulin complex is part of a cellular mechanism for regulating the levels and dimerization of tubulin chains.

FIG. 1

The His₆-Rbl2p–β-tubulin complex formed in vivo is devoid of α-tubulin. LTY292 cells carrying a plasmid-borne gene specifying His₆-Rbl2p under control of the GAL promoter were grown for 2 h in medium containing raffinose (uninduced) or galactose (induced). The His₆-Rbl2p was separated from extracts by using Ni-NTA beads. The whole-cell extracts (W.C.E.) and bound proteins were analyzed by immunoblotting for β-tubulin, α-tubulin, and Rbl2p as shown. The bottom film was exposed six times longer than the top two. The presence of β-tubulin in the bound proteins required expression of His₆-Rbl2p, but no α-tubulin was detected in the bound proteins under either condition.

FIG. 2

Newly synthesized β-tubulin can bind to Rbl2p in vivo. Distribution of β-tubulin between the Rbl2p and α-tubulin pools in FSY 820 cells is shown. (A) Immunoblots of whole-cell extracts (W.C.E.), anti-α-tubulin immunoprecipitates (I.P.), and proteins bound to His₆-Rbl2p either at steady state (s.s.) or after a brief (galactose for 10 min followed by glucose for 10 min) induction of Tub2-590p (pulse) are shown. The blots were probed with antibodies specific for either wild-type Tub2p (206) or the faster-migrating Tub2-590p (339). Sample volumes and exposure times were adjusted to give detectable signals from all fractions. (B) Analysis of the data shown in panel A. The ratios represent the proportions of the induced β-tubulin (Tub2-590p) relative to the constitutive β-tubulin (Tub2p) present as the α/β-tubulin heterodimer or associated with Rbl2p from uninduced cells (steady state) and after a brief induction of Tub2-590p expression (pulse).

FIG. 3

Formation of His₆-Rbl2p–β-tubulin complex in vitro. Bacterial extract containing His₆-Rbl2p was incubated with wild-type yeast cell extracts at 4°C. At various times, an aliquot was removed and added to Ni-NTA beads for 15 min. The beads were washed, and the bound proteins were assayed by elution followed by immunoblotting with anti-tubulin antibodies. The data are reported as percent β-tubulin and α-tubulin bound as a function of time.

FIG. 4

Rbl2p does not bind to denatured β-tubulin. Extracts from wild-type cells (W.C.E.) were incubated either with control buffer (−) or with 6 M guanidine hydrochloride (+) for 5 min, diluted 100-fold, and then incubated with His₆-Rbl2p plus Ni-NTA beads. The specifically bound proteins were eluted from the beads and assayed for the presence of both β-tubulin and α-tubulin by immunoblotting. The binding of β-tubulin to Rbl2p was essentially abolished by the preincubation with denaturing agent. No bound α-tubulin was detected under either condition.

FIG. 5

Dissociation of His₆-Rbl2p–β-tubulin and α-His₆–β-tubulin in vitro. The dissociation rates of these two complexes were measured by incubating cell extracts with Ni-NTA beads, resuspending the beads in PME buffer at 4°C, and then measuring the levels of the complexes remaining at various times by immunoblotting. The data are reported as semi-log plots of β-tubulin (circles) and α-tubulin (squares) dissociation. Extracts from cells expressing either Tub2p and His₆-Rbl2p or His₆-Tub2p alone were used to measure the dissociation of β-tubulin or α-tubulin, respectively.