Cells lacking the minor α-tubulin gene, TUB3, require RBL2 but not CIN1 for viability. (A) Serial dilutions of cells with both RBL2 and TUB3 deleted and carrying a low-copy TUB3 plasmid marked by URA3 were plated to synthetic complete medium (SC) and to synthetic complete medium containing 5-FOA (SC 5-FOA), which selects against retention of plasmids expressing URA3. Cells that contain pRBL2-HIS3-CEN (+) can grow on both media, but cells without that plasmid (−) cannot grow on 5-FOA. (B) Serial dilutions of cells with both CIN1 and TUB3 deleted and carrying a low-copy TUB3 plasmid marked by URA3 were plated to synthetic complete medium (SC) and to synthetic complete medium containing 5-FOA (SC 5-FOA). The cin1Δ tub3Δ double mutants can survive in the absence of an extra plasmid copy of TUB3.

Quantitation of Rbl2p and β-tubulin in wild-type cells. (A) Immunoblots with anti-Rbl2p antibody of extracts of a known number of wild-type cells and of purified Rbl2p standards. (B) Immunoblots with anti-β-tubulin antibody of extracts of a known number of wild-type cells and of purified β-tubulin standards. (C) Immunoblots with anti-β-tubulin, anti-α-tubulin, and anti-Rbl2p antibodies of extracts from a known number of wild-type (WT), tub3Δ, and pac10Δ cells. The data shown are representative of at least three independent experiments.

An extra genomic copy of RBL2 is required to rescue cells from the synthetic lethality of tub3Δ pac10Δ. The inviability of cells with PAC10 and TUB3 deleted is suppressed by a plasmid expressing RBL2 (Fig. 1). SC, synthetic complete medium.

Colonies similar in size formed by cells that survive β-tubulin overexpression. Cells containing an integrated copy of TUB2 under the control of the galactose promoter and a range of RBL2 gene dosages were plated to both glucose and galactose plates. The percentage of the cells able to form colonies on galactose is indicated in parentheses (Table 2). The colonies formed by the four strains are essentially identical in size, although there is some size variability within all four strains.

Levels of pRBL2-CEN and Rbl2p expression are upregulated in cells surviving β-tubulin overexpression. FSY583 cells containing a low-copy plasmid expressing RBL2 and an integrated copy of GAL-TUB2 were grown on glucose and galactose. DNA preparations from these cells were analyzed by Southern blotting to determine the level of the RBL2 sequence (A), and cell extracts were analyzed by immunoblotting to determine the level of Rbl2p (B) (see Materials and Methods).

Rbl2p does not sequester free β-tubulin into an Rbl2p/β-tubulin complex. Total protein was prepared from four different strains and analyzed by Sephacryl S-300 HR gel filtration chromatography. Each fraction was assayed for α-tubulin, β-tubulin, and Rbl2p by immunoblotting, followed by scanning densitometry. The graphs plot the percentage of the total amount of each protein in each fraction and illustrate the elution positions of β-tubulin (diamonds), α-tubulin (circles), and Rbl2p (triangles). All of the experiments were repeated at least three times. Panels: A, wild-type cells; B, cells overexpressing both β-tubulin and Rbl2p; C, tub1-724 cells overexpressing Rbl2p; D, cells overexpressing β- and α-tubulin; E, tub3Δ cells; E, cells overexpressing β-tubulin. V, void volume; β, expected position of a peak of monomeric β-tubulin.

Ability of aggregates to protect cells from β-tubulin overexpression. (A) Cells containing a single integrated copy of GAL-TUB2 and a plasmid copy of RBL2 under the control of the MET25 promoter were grown under inducing conditions for 3 h to allow an aggregate to form. The cells were then shifted to medium containing methionine to repress Rbl2p expression. Cells containing an aggregate preformed in the presence of Rbl2p (triangles) died from β-tubulin overexpression with the same kinetics as cells only overexpressing β-tubulin (squares). As a positive control, we included cells in which both Rbl2p and β-tubulin were overexpressed for the duration of the experiment (circles). (B) Cells containing an integrated copy of GAL-TUB2 with or without an extra genomic copy of RBL2 were transformed with either control plasmid pGAL-TUB2-STOP or plasmid pGAL-TUB2-3′3/4 overexpressing the C-terminal three-fourths of TUB2. The C-terminal fragment forms β-tubulin aggregates when overexpressed (data not shown). The pGAL-TUB2-STOP control plasmid does not affect the ability of cells to survive β-tubulin overexpression; pRBL2 alone is sufficient to allow >1% of the cells to survive β-tubulin overexpression (see Table 2). The pGAL-TUB2-STOP plasmid serves as a control for the reduction of β-tubulin overexpression that can occur when a second galactose-inducible promoter is introduced into cells. Although the aggregate formed by the β-tubulin fragment is a weak suppressor of β-tubulin overexpression, it enhances the suppression by RBL2. The data shown are representative of three independent experiments.

A model for Rbl2p function in vivo. Rbl2p protects cells by binding transiently to free β-tubulin. Once released from Rbl2p, free β-tubulin has three possible fates: it can bind to the target of β-tubulin toxicity, be sequestered in the aggregate, or reassociate with Rbl2p. To protect cells from free β-tubulin, the level of Rbl2p in the cells must reach a threshold level so that Rbl2p can effectively compete with the target of β-tubulin toxicity for β-tubulin binding. Associations of β-tubulin with both the target of β-tubulin toxicity and the aggregate of β-tubulin appear to be irreversible. Rbl2p may facilitate aggregate formation by converting β-tubulin into a form that aggregates more readily.