Regulation of tubulin functions in Saccharomyces cerevisiae

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.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2001

Author dc:creator

• Abruzzi, Katharine Boyer Compton, 1973-

Advisor dc:contributor.advisor

• Frank Solomon.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/8584

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/8584