A GTP:AMP phosphotransferase, Adk2p, in Saccharomyces cerevisiae. Role of the C terminus in protein folding/stabilization, thermal tolerance, and enzymatic activity.
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 degrees 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.
Gu
Yajuan
Y
Department of Pharmacology and Physiology, UMDNJ-New Jersey Medical School, Newark, New Jersey 07103-1709, USA.
Gordon
Donna M
DM
Amutha
Boominathan
B
Pain
Debkumar
D
D000263:Adenylate Kinase / Q000737:chemistry*; D000262:Adenylyl Cyclases / Q000737:chemistry* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003593:Cytoplasm / Q000378:metabolism; D017353:Gene Deletion; D006358:Hot Temperature; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013696:Temperature; D014357:Trypsin / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology
D029701:Saccharomyces cerevisiae Proteins; D014508:Urea; D000263:Adenylate Kinase; D014357:Trypsin; D000262:Adenylyl Cyclases; C501854:Adk2 protein, S cerevisiae