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© The Rockefeller University Press, 0021-9525/1999//113 $5.00
The Journal of Cell Biology, Volume 144, Number 1, , 1999 113-124

Article

Alf1p, a CLIP-170 Domain-containing Protein, Is Functionally and Physically Associated with {alpha}-Tubulin



Becket Feierbach*, Eva Nogales{ddagger},§, Kenneth H. Downing{ddagger}, and Tim Stearns*

* Department of Biological Sciences, Stanford University, Stanford, California 94305-5020; {ddagger} Life Science Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720; and § Molecular and Cell Biology Department, University of California, Berkeley, California 94720

Tubulin is a heterodimer of {alpha}- and β-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287–296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821–832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with {alpha}-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with {alpha}-tubulin. Mutations in {alpha}-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of {alpha}-tubulin; this domain is distinct from the region of interaction between {alpha}-tubulin and β-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in {alpha}-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast {alpha}-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional {alpha}-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester {alpha}-tubulin from interaction with β-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

Key Words: tubulin • microtubule • Saccharomyces cerevisiae • chaperonin • CLIP-170

Abbreviations used in this paper: c-cpn, cytosolic chaperonin; GFP, green fluorescent protein; SD, synthetic dextrose; YEP, yeast extract/peptone; SD, synthetic dextrose.

Address correspondence to Tim Stearns, Department of Biological Sciences, Stanford University, Stanford, CA 94305-5020. Tel.: (650) 725-6934. Fax: (650) 725-8309. E-mail: stearns{at}stanford.edu


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