title: Structural and enzymatic properties of the AAA protein Drg1p from Saccharomyces cerevisiae. Decoupling of intracellular function from ATPase activity and hexamerization.
abstract: The AAA protein Drg1 from yeast was affinity-purified, and its ATPase activity and hexamerization properties were analyzed. The same parameters were also determined for several mutant proteins and compared in light of the growth characteristics of the corresponding cells. The protein from a thermosensitive mutant exhibited reduced ATPase activity and hexamerization. These defects were not reversed by an intragenic suppressor mutation, although this allele supported growth at the nonpermissive temperature. A different set of mutants was generated by site-specific mutagenesis intended to adjust the Walker A box of the D2 domain of Drg1p to that of the D1 domain. A S562G exchange in D2 produced a nonfunctional protein that did not hexamerize but showed above-normal ATPase activity. The C561T mutant protein, on the other hand, was functional but hexamerized less readily and had reduced ATPase activity. In contrast, the C561T/S562G protein hexamerized less than wild type but had much higher ATPase activity. We distinguished strong and weak ATP-binding sites in the wild type protein but two weak sites in the C561T/S562G protein, indicating that the stronger site resides in D2. These observations are discussed in terms of the inter-relationship of ATPase activity per se, oligomeric status, and intracellular function for AAA proteins.
authors: Institut für Molekularbiologie, Biochemie und Mikrobiologie, Karl-Franzens-Universität Graz, Universitätsplatz 2, A-8010 Graz, Austria.
authors: Andriy
authors: A
authors: Zakalskiy
authors: Gregor
authors: G
authors: Högenauer
authors: Takashi
authors: T
authors: Ishikawa
authors: Eva
authors: E
authors: Wehrschütz-Sigl
authors: Franz
authors: F
authors: Wendler
authors: David
authors: D
authors: Teis
authors: Gertrude
authors: G
authors: Zisser
authors: Alasdair C
authors: AC
authors: Steven
authors: Helmut
authors: H
authors: Bergler
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000483:Alleles; D001665:Binding Sites; D001896:Boron Compounds / Q000494:pharmacology; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008854:Microscopy, Electron; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
chemical_list: D001896:Boron Compounds; D005656:Fungal Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C463962:AFG2 protein, S cerevisiae; D005990:Glycerol