Protection from Free β-Tubulin by the β-Tubulin Binding Protein Rbl2p

Abstract

Free ␤-tubulin not in heterodimers with ␣-tubulin can be toxic, disrupting microtubule assembly and function. We are interested in the mechanisms by which cells protect themselves from free ␤-tubulin. This study focused specifically on the function of Rbl2p, which, like ␣-tubulin, can rescue cells from free ␤-tubulin. In vitro studies of the mammalian homolog of Rbl2p, cofactor A, have suggested that Rbl2p/cofactor A may be involved in tubulin folding. Here we show that Rbl2p becomes essential in cells containing a modest excess of ␤-tubulin relative to ␣-tubulin. However, this essential activity of Rbl2p/cofactorA does not depend upon the reactions described by the in vitro assay. Rescue of ␤-tubulin toxicity requires a minimal but substoichiometric ratio of Rbl2p to ␤-tubulin. The data suggest that Rbl2p binds transiently to free ␤-tubulin, which then passes into an aggregated form that is not toxic.Studies of cellular control of microtubule assembly have focused primarily on the assembly reaction from ␣/␤-tubulin heterodimers to microtubule polymers and on the identification of protein cofactors and structures that modulate this polymerization (8-10). 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 ␣-and ␤-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 ␤-tubulin (5), Rbl2p binds monomeric ␤-tubulin to form a heterodimer that excludes ␣-tubulin, both in vivo and in vitro (5). Pulse-labeling experiments demonstrate that Rbl2p can bind both newly synthesized ␤-tubulin before it is incorporated into ␣/␤-tubulin heterodimers and ␤-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 ␣/␤-tubulin heterodimers (14, 30). 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 ␤-tubulin polypeptides are released from the cytosolic chaperonin, they are able initially to bind either cofactor A or c...

PhLP3 Modulates CCT-mediated Actin and Tubulin Folding via Ternary Complexes with Substrates

“…7B). This is consistent with the different effects of PFD and cofactor deletions on the ratios of ␣-and ␤-tubulin; PLP1 deletion corrects the excess of quasi-native ␤-tubulin responsible for benomyl sensitivity in ⌬pac10 cells, although it has no observable effect on cofactor deleted cells, which have a normal ratio of ␣-to ␤-tubulin (46,47).…”

Consequences of Defective Tubulin Folding on Heterodimer Levels, Mitosis and Spindle Morphology in Saccharomyces cerevisiae

“…Gel filtration chromatography was performed as described previously (Abruzzi et al 2002). Briefly, cell extracts were obtained through French Press cell lysis and applied to a Sephacryl S-300HR column.…”

“…Immunoblot detection was performed as previously described (Abruzzi et al 2002), and by using the ECL Plus chemiluminescence reagent kit (Amersham Biosciences, Arlington Heights, IL) and fluorescence imaging (Storm System; Molecular Dynamics, Sunnyvale, CA), and quantitated by ImageQuant Macintosh software. Western blots were repeated at least four times and results were averaged.…”

Revisiting the tubulin folding pathway: new roles in centrosomes and cilia

“…However, when these proteins started to be studied in mammalian models, it became clear that they play crucial functions, not always directly related to their expected role in the tubulin folding pathway but still related to the cytoskeleton. For example, TBCA, which in yeast has been proposed to function as a reservoir of excess of b-tubulin, is dispensable for tubulin folding in vitro (53,54). Nevertheless, TBCA is an essential gene in human cell lines with a preponderant role in the recycling of mature tubulin heterodimers (54) (Nolasco et al, unpublished).…”