title: Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin.
abstract: Tubulin is a heterodimer of alpha- and beta-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 beta-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 beta-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.
authors.affiliation: Department of Biological Sciences, Stanford University, Stanford, California 94305-5020, USA.
authors.forename: B
authors.initials: B
authors.lastname: Feierbach
authors.forename: E
authors.initials: E
authors.lastname: Nogales
authors.forename: K H
authors.initials: KH
authors.lastname: Downing
authors.forename: T
authors.initials: T
authors.lastname: Stearns
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002874:Chromosome Mapping; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; C097366:PAC2 protein, S pombe; D011993:Recombinant Fusion Proteins; D029702:Schizosaccharomyces pombe Proteins; D014157:Transcription Factors; D014404:Tubulin; C076744:cytoplasmic linker protein 170