Regulation of tubulin functions in Saccharomyces cerevisiae
Author(s)Abruzzi, Katharine Boyer Compton, 1973-
Massachusetts Institute of Technology. Dept. of Biology.
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A variety of cellular factors regulate the formation of complex microtubule structures in the cell. Most of these factors modulate microtubule structures by affecting the polymerization of [alpha]/[beta]-tubulin heterodimer subunits to form microtubules. However, there is another class of factors that affect microtubule formation by regulating the steps that precede the polymerization of [alpha]/[beta]-tubulin heterodimer. One of these factors is Rbl2p. Rbl2p was originally isolated in a screen for genes that when overexpressed can rescue the lethality of [beta]-tubulin overexpression. The mammalian homolog of Rbl2p, Cofactor A, was isolated as a non-essential cofactor in an in vitro tubulin folding reaction (Tian et al., 1996). Both Rbl2p and Cofactor A form a heterodimer with monomeric [beta]-tubulin that excludes [beta]-tubulin (Archer et al., 1995; Melki et al., 1996). The assays that identified Rbl2p and its homolog Cofactor A suggest two possible models to explain the ability of Rbl2p/CofactorA to protect cells from free [beta]-tubulin in vivo. First if unfolded [beta]-tubulin is toxic, Rbl2p could protect cells from free [beta]-tubulin by catalyzing the folding of [beta]-tubulin1 as proposed in the in vitro tubulin folding assay. Second, Rbl2p could bind to and sequester free [beta]-tubulin into a Rbl2p/[beta]-tubulin heterodimer. Our data suggest that Rbl2p's in vivo function is more complicated than predicted by either of these two models. Rbl2p binds transiently to a subpopulation of the free [beta]-tubulin and prevents it from interacting with the target of [beta]-tubulin toxicity until it associates with an aggregate of [beta]-tubulin. Cells expressing an allele of [alpha]-tubulin, tubl-729, arrest in the cold as largebudded cells with either short spindles or no microtubules. The spindle defect checkpoint proteins Bublp and Bub3p suppress the cold-sensitivity of tubl-729 cells (Guenette et al., 1995). We tested whether other proteins involved in the mitotic checkpoints can suppress tubl-729 cells. Our results show that an extra genomic copy of MPSl also suppresses the cold-sensitivity of tubl-729 cells, but that genomic copies of BUB2 and MADsl-3 do not. Bub3p rescues the coldsensitivity of tubl-729 cells in which deletion of MAD2 has eliminated the spindle defect checkpoint. This suggests that Bub3p does not act through the spindle defect checkpoint to suppress tubl-729 cells. We present preliminary evidence in support of a model proposing that tubl-729 cells are defective in kinetochoremicrotubule attachment and that Bublp, Bub3p, and Mpslp may suppress the phenotypes of tubl-729 cells by strengthening these attachments.
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2001.Includes bibliographical references.
DepartmentMassachusetts Institute of Technology. Dept. of Biology.
Massachusetts Institute of Technology