Alf1p, a CLIP-170 domain-containing protein, is functionally and physically associated with alpha-tubulin

J Cell Biol. 1999 Jan 11;144(1):113-24. doi: 10.1083/jcb.144.1.113.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Chromosome Mapping
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Fungal Proteins / genetics
  • Fungal Proteins / metabolism*
  • Gene Expression
  • Microtubule-Associated Proteins / genetics
  • Microtubule-Associated Proteins / metabolism*
  • Microtubules / metabolism
  • Molecular Sequence Data
  • Neoplasm Proteins
  • Phenotype
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Schizosaccharomyces pombe Proteins*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism*
  • Tubulin / metabolism*

Substances

  • DNA-Binding Proteins
  • Fungal Proteins
  • Microtubule-Associated Proteins
  • Neoplasm Proteins
  • PAC2 protein, S pombe
  • Recombinant Fusion Proteins
  • Schizosaccharomyces pombe Proteins
  • Transcription Factors
  • Tubulin
  • cytoplasmic linker protein 170