A purified subfragment of yeast Atp11p retains full molecular chaperone activity

J Biol Chem. 2003 Sep 5;278(36):34110-3. doi: 10.1074/jbc.M305353200. Epub 2003 Jun 26.

Abstract

Atp11p is a molecular chaperone of the mitochondrial matrix that participates in the biogenesis pathway to form F1, the catalytic unit of the ATP synthase. Affinity tag pull-down assays and yeast two-hybrid screens have shown that Atp11p binds to free beta subunits of F1 (Wang, Z. G., and Ackerman, S. H. (2000) J. Biol. Chem. 275, 5767-5772). This binding action prevents the beta subunit from associating with itself in non-productive complexes and fosters the formation of a (alpha beta)3 hexamer. Following the premise that Atp11p action is mediated primarily through a surface (as opposed to specific amino acids, as in an enzyme active site), solving its three-dimensional structure so that we may learn how the shape of the protein influences its function is a high priority. Recombinant yeast Atp11p has proven refractory for such analysis because of the presence of a disordered region in the protein. In this article, we show that removal of 67 residues from the amino terminus of recombinant Atp11p yields a subfragment of the protein (called Atp11pTRNC) that retains molecular chaperone function as determined in vitro with both a surrogate substrate (reduced insulin) and the natural substrate (F1 beta). Moreover, preliminary 15N-1H heteronuclear single quantum coherence spectra obtained with Atp11pTRNC indicate that the truncated protein is well ordered and amenable to structure determination by nuclear magnetic resonance.

Publication types

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

MeSH terms

  • Binding Sites
  • Catalysis
  • Electrophoresis, Polyacrylamide Gel
  • Hydrogen-Ion Concentration
  • Magnetic Resonance Spectroscopy
  • Models, Genetic
  • Molecular Chaperones / chemistry*
  • Molecular Chaperones / metabolism*
  • Plasmids / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Proton-Translocating ATPases / chemistry*
  • Proton-Translocating ATPases / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / enzymology
  • Schizosaccharomyces pombe Proteins / chemistry*
  • Schizosaccharomyces pombe Proteins / metabolism
  • Time Factors

Substances

  • Atp11 protein, S pombe
  • Molecular Chaperones
  • Recombinant Proteins
  • Schizosaccharomyces pombe Proteins
  • Proton-Translocating ATPases