Functional characterization and localization of Pneumocystis carinii lanosterol synthase.
Organisms in the genus Pneumocystis are ubiquitous, opportunistic pathogenic fungi capable of causing a lethal pneumonia in immunocompromised mammalian hosts. Pneumocystis spp. are unique members of the fungal kingdom due to the absence of ergosterol in their cellular membranes. Although these organisms were thought to obtain cholesterol by scavenging, transcriptional analyses indicate that Pneumocystis carinii encodes gene homologs involved in sterol biosynthesis. To better understand the sterol pathway in these uncultivable fungi, yeast deletion strains were used to interrogate the function and localization of P. carinii lanosterol synthase (ERG7). The expression of PcErg7p in an ERG7-null mutant of the yeast Saccharomyces cerevisiae did not alter its growth rate and produced a functional lanosterol synthase, as evidenced by the presence of lanosterol detected by gas chromatographic analysis in levels comparable to that produced by the yeast enzyme. Western blotting and fluorescence microscopy revealed that, like the S. cerevisiae Erg7p, the PcErg7p localized to lipid particles in yeast. Using fluorescence microscopy, we show for the first time the presence of apparent lipid particles in P. carinii and the localization of PcErg7p to lipid particles in P. carinii. The detection of lipid particles in P. carinii and their association with PcErg7p therein provide strong evidence that the enzyme serves a similar function in P. carinii. Moreover, the yeast heterologous system should be a useful tool for further analysis of the P. carinii sterol pathway.
Joffrion
Tiffany M
TM
Department of Internal Medicine, University of Cincinnati College of Medicine, 231 Albert Sabin Way, Cincinnati, OH 45267-0560, USA. joffritm@mail.uc.edu
Collins
Margaret S
MS
Sesterhenn
Thomas
T
Cushion
Melanie T
MT
D000595:Amino Acid Sequence; D000818:Animals; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D019751:Intramolecular Transferases / Q000235:genetics / Q000378:metabolism*; D050356:Lipid Metabolism / Q000235:genetics; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism; D045363:Pneumocystis carinii / Q000166:cytology / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013261:Sterols / Q000096:biosynthesis
D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D013261:Sterols; D019751:Intramolecular Transferases; C021924:lanosterol synthase