Activation of Archaeoglobus fulgidus Cu+-ATPase CopA by cysteine

AbstractCopA, a thermophilic ATPase from Archaeoglobus fulgidus, drives the outward movement of Cu+ across the cell membrane. Millimolar concentration of Cys dramatically increases (≅800%) the activity of CopA and other PIB-type ATPases (Escherichia coli ZntA and Arabidopsis thaliana HMA2). The high affinity of CopA for metal (≅1 μM) together with the low Cu+–Cys KD (<10−10M) suggested a multifaceted interaction of Cys with CopA, perhaps acting as a substitute for the Cu+ chaperone protein present in vivo. To explain the activation by the amino acid and further understand the mechanism of metal delivery to transport ATPases, Cys effects on the turnover and partial reactions of CopA were studied. 2–20 mM Cys accelerates enzyme turnover with little effect on CopA affinity for Cu+, suggesting a metal independent activation. Furthermore, Cys activates the p-nitrophenyl phosphatase activity of CopA, even though this activity is metal independent. Cys accelerates enzyme phosphorylation and the forward dephosphorylation rates yielding higher steady state phosphoenzyme levels. The faster dephosphorylation would explain the higher enzyme turnover in the presence of Cys. The amino acid has no significant effect on low affinity ATP Km suggesting no changes in the E1↔E2 equilibrium. Characterization of Cu+ transport into sealed vesicles indicates that Cys acts on the cytoplasmic side of the enzyme. However, the Cys activation of truncated CopA lacking the N-terminal metal binding domain (N-MBD) indicates that activation by Cys is independent of the regulatory N-MBD. These results suggest that Cys is a non-essential activator of CopA, interacting with the cytoplasmic side of the enzyme while this is in an E1 form. Interestingly, these effects also point out that Cu+ can reach the cytoplasmic opening of the access path into the transmembrane transport sites either as a free metal or a Cu+–Cys complex

Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation

Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases (∼50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis

Hsp110 Chaperones Control Client Fate Determination in the Hsp70–Hsp90 Chaperone System

The Hsp110 family of protein chaperones was known to promote maturation of Hsp90 client proteins. The yeast Hsp110 ortholog Sse1 is now shown to influence the decision to fold or degrade substrates of the Hsp70–Hsp90 chaperone system when maturation is compromised

Interaction of KH16p with 14-3-3ζ using ¹H NMR spectroscopy.

<p>¹H NMR spectra of the KH16p peptide with an increasing concentration of 14-3-3ζ demonstrating the linewidth broadening in the amide, aromatic, and aliphatic regions. The ¹H NMR experiments were carried out in aqueous solution on a Bruker AVANCE III 500MHz spectrometer at 288 K.</p

STD NMR study of KH16p and KH16 with 14-3-3ζ.

<p><b>(A)</b> One-dimensional ¹H NMR reference spectrum of KH16p in the presence of 14-3-3ζ at 500:1 molar ratio. <b>(B)</b> The absence of STD signals in the aromatic region and the prominent STD signals in the aliphatic region demonstrate the close proximity of few residues of KH16p to the 14-3-3ζ protein. <b>(C)</b> One-dimensional ¹H NMR reference spectrum of KH16 in the presence of 14-3-3ζ at 500:1 molar ratio. <b>(D)</b> STD spectrum of KH16. The STD NMR experiments were carried out in aqueous solution on a Bruker AVANCE III 500 MHz spectrometer at 288 K.</p