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J. Biol. Chem., Vol. 280, Issue 19, 18604-18609, May 13, 2005

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A GTP:AMP Phosphotransferase, Adk2p, in Saccharomyces cerevisiae

ROLE OF THE C TERMINUS IN PROTEIN FOLDING/STABILIZATION, THERMAL TOLERANCE, AND ENZYMATIC ACTIVITY^*

Yajuan Gu, Donna M. Gordon, Boominathan Amutha, and Debkumar Pain

From the Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103

Adenylate kinases participate in maintaining the homeostasis of cellular nucleotides. Depending on the yeast strains, the GTP:AMP phosphotransferase is encoded by the nuclear gene ADK2 with or without a single base pair deletion/insertion near the 3' end of the open reading frame, and the corresponding protein exists as either Adk2p (short) or Adk2p (long) in the mitochondrial matrix. These two forms are identical except that the three C-terminal residues of Adk2p (short) are changed in Adk2p (long), and the latter contains an additional nine amino acids at the C terminus of the protein. The short form of Adk2p has so far been considered to be inactive (Schricker, R., Magdolen, V., Strobel, G., Bogengruber, E., Breitenbach, M., and Bandlow, W. (1995) J. Biol. Chem. 270, 31103–31110). Using purified proteins, we show that at the physiological temperature for yeast growth (30 °C), both short and long forms of Adk2p are enzymatically active. However, in contrast to the short form, Adk2p (long) is quite resistant to thermal inactivation, urea denaturation, and degradation by trypsin. Unfolding of the long form by high concentrations of urea greatly stimulated its import into isolated mitochondria. Using an integration-based gene-swapping approach, we found that regardless of the yeast strains used, the steady state levels of endogenous Adk2p (long) in mitochondria were 5–10-fold lower compared with those of Adk2p (short). Together, these results suggest that the modified C-terminal domain in Adk2p (long) is not essential for enzyme activity, but it contributes to and strengthens protein folding and/or stability and is particularly important for maintaining enzyme activity under stress conditions.

Received for publication, January 24, 2005 , and in revised form, March 4, 2005.

^* This work was supported by American Heart Association Grants 0335473T (to D. G.) and 0355710T (to D. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY949617.

To whom correspondence should be addressed: Dept. of Pharmacology and Physiology, New Jersey Medical School, University of Medicine and Dentistry, 185 S. Orange Ave., MSB I-669, Newark, NJ 07103-1709. Tel.: 973-972-3439; Fax: 973-972-4554; E-mail: painde{at}umdnj.edu.

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