Structural and Enzymatic Properties of the AAA Protein Drg1p fromSaccharomyces cerevisiae

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.

A conserved inter-domain communication mechanism regulates the ATPase activity of the AAA-protein Drg1

“…S3). These values are in good agreement with earlier estimates obtained by radioactive nucleotide binding studies with wildtype Drg127. With respect to ADP binding we measured a K d(ADP) of about 120 μM for the D1 domain, while binding to D2 was below the detection limits of our experimental setup.…”

“…27. These constructs contain a uracil auxotrophy marker, the copper-inducible CUP1 -promoter and an N-terminal GST-tag, which is separated from the DRG1 -alleles by a PreScission TM protease (GE Healthcare) site to allow removal of the GST-tag.…”

“…Drg1 (Diazaborine resistance gene 1) is an essential type II AAA-ATPase in the yeast Saccharomyces cerevisiae and is highly related to mammalian p97 and its yeast orthologue Cdc4827. In previous works we have characterized Drg1 as a key player of eukaryotic ribosome biogenesis that initiates the final maturation of the large ribosomal 60S-subunit in the cytoplasm82829.…”

Characterization and evolutionary history of an archaeal kinase involved in selenocysteinyl-tRNA formation

“…ATPase activity was determined by measuring the amount of [α- 32 P]ATP converted to [α- 32 P]ADP as described before with modifications (18). These assays were carried out in a 5 µl reaction volume including 1× PSTK buffer with 130 nM cold ATP, 100 nM [α- 32 P]ATP and 200 nM enzyme at 37°C for 45 min.…”

ANCCA, an estrogen-regulated AAA+ ATPase coactivator for ERα, is required for coregulator occupancy and chromatin modification

“…Protein samples were then separated by SDS/PAGE and processed for autoradiography. ATP hydrolysis assay was performed as previously described with modifications (47). Briefly, Ϸ0.5 g of ANCCA or control proteins was mixed in 15 l of buffer containing 17 mM Tris⅐Cl (pH 8.0), 100 mM NaCl, 10 mM MgCl 2 , 0.3 mM PMSF, 0.3 mM DTT, and 5% glycerol.…”