Abstract
The Thermoplasma VCP-like ATPase from Thermoplasma acidophilum (VAT) ATPase is a member of the two-domain AAA ATPases and homologous to the mammalian p97/VCP and NSF proteins. We show here that the VAT ATPase complex unfolds green fluorescent protein (GFP) labeled with the ssrA-degradation tag. Increasing the Mg2+ concentration derepresses the ATPase activity and concomitantly stimulates the unfolding activity of VAT. Similarly, the VATDeltaN complex, a mutant of VAT deleted for the N domain, displays up to 24-fold enhanced ATP hydrolysis and 250-fold enhanced GFP unfolding activity when compared with wild-type VAT. To determine the individual contribution of the two AAA domains to ATP hydrolysis and GFP unfolding we performed extensive site-directed mutagenesis of the Walker A, Walker B, sensor-1, and pore residues in both AAA domains. Analysis of the VAT mutant proteins, where ATP hydrolysis was confined to a single AAA domain, revealed that the first domain (D1) is sufficient to exert GFP unfolding indistinguishable from wild-type VAT, while the second AAA domain (D2), although active, is significantly less efficient than wild-type VAT. A single conserved aromatic residue in the D1 section of the pore was found to be essential for GFP unfolding. In contrast, two neighboring residues in the D2 section of the pore had to be exchanged simultaneously, to achieve a drastic inhibition of GFP unfolding.
Highlights
VCP proteins belong to the latter family and are built of two adjacent AAA domains and were originally suggested to function in membrane fusion [3]
VCP-like ATPase from Thermoplasma acidophilum (VAT) Shows Mg2ϩ-stimulated Unfolding of GFP11—In search for an assay to demonstrate unfolding activity of VAT directly, we employed GFP11, a C-terminal fusion of the globular green fluorescent protein with the 11-residue peptide ssrA [15]
The substrate did not account for the increase of the ATP hydrolysis by VAT, since neither the ATPase activity of VAT nor VAT⌬N was stimulated by GFP11
Summary
Site-directed Mutagenesis—To generate the mutant variants of VAT, codons for Lys237 and/or Lys514 (Walker A), Glu291 and/or Glu568 (Walker B), Asn334 and/or Asn612 (sensor-1), Tyr264 and/or Trp541, and/or Val542 (pore residues) in plasmid pET-28-VAT-His containing the codon optimized vat gene [14] were changed to encode alanine. The final protein solution was purified on a Superose-6 column (Amersham Biosciences) equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl. Thawed cells containing VAT-K237/514A were suspended in 20 mM HEPES, pH 7.5, 300 mM NaCl, and 10 mM imidazole and processed as described above. The eluent was purified on a Superose-6 column (Amersham Biosciences) equilibrated with 20 mM HEPES, pH 7.5, 150 mM NaCl. VAT⌬N cells were suspended in 20 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM dithiothreitol, and processed as described above. The fractions containing the VAT⌬N protein were pooled, loaded onto nickel-nitrilotriacetic acid beads (Qiagen), washed with 20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM MgCl2, 20 mM imidazole, and eluted with 500 mM imidazole in the same buffer. Western Blot Analysis—The Western blot analysis of GFP11 degradation by proteasomes was performed with anti-enhanced cyan fluorescent protein (a mutant of GFP) antibodies
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
