The Pneumocystis meiotic PCRan1p kinase exhibits unique temperature-regulated activity.
Pneumocystis organisms are opportunistic fungal pathogens that cause significant pneumonia in immune-compromised hosts. Recent evidence has suggested that Pneumocystis carinii exists as separate mating types, and expresses and regulates proteins that govern meiosis and progression of the life cycle. This study was undertaken to investigate the activity of three life cycle-regulatory proteins in Pneumocystis, including two proteins essential in mating signaling, and a putative meiotic regulator, to determine the conditions under which they are most active. This study used V5/HIS-tagged PCRan1p, PCSte20p, and PCCbk1, purified from Saccharomyces cerevisiae strain, INVSC, as well as an in vitro Escherichia coli protein expression system to determine the optimal expression conditions of each protein in the presence of varying pH, temperature, and metal ions. These studies demonstrate an atypical enzymatic activity in PCRan1p, whereby the kinase was most active in the environmental conditions between 10 and 25 degrees C, compared with a dramatic reduction in activity above 30 degrees C, temperatures typically found within mammalian hosts. Circular dichroism and fluorescence spectroscopy suggest that PCRan1p becomes partially unfolded at 25 degrees C, leading to its most active conformation, whereas continued unfolding as temperature increases results in strongly suppressed activity. These studies suggest that, in vivo, while under conditions within the mammalian lung (typically 37 degrees C), PCRan1p kinase activity is largely suppressed, allowing better conditions for the activation of meiosis, whereas in ex vivo environments, PCRan1p kinase activity increases to arrest progression of the life cycle until conditions become more favorable.
Burgess
Joshua W
JW
Thoracic Disease Research Unit, 8-24 Stabile Building, Mayo Clinic, 200 1st Street SW, Rochester, MN 55905, USA.
Kottom
Theodore J
TJ
Villegas
Leah R
LR
Lamont
Jeffrey D
JD
Baden
Elizabeth M
EM
Ramirez-Alvarado
Marina
M
Limper
Andrew H
AH
D001483:Base Sequence; D002942:Circular Dichroism; D017931:DNA Primers / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008540:Meiosis; D045363:Pneumocystis carinii / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D011489:Protein Denaturation; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000378:metabolism; D013045:Species Specificity; D013050:Spectrometry, Fluorescence; D013696:Temperature
D017931:DNA Primers; D005656:Fungal Proteins; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D011494:Protein Kinases; C087019:ran1 protein, S pombe; D017346:Protein Serine-Threonine Kinases