FEMS yeast research, 2013-05, Vol.13 (3), p.302-311
2013
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- Autor(en) / Beteiligte
- Titel
- Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae
- Ist Teil von
- FEMS yeast research, 2013-05, Vol.13 (3), p.302-311
- Ort / Verlag
- Oxford, UK: Blackwell Publishing Ltd
- Erscheinungsjahr
- 2013
- Quelle
- Wiley Online Library Journals Frontfile Complete
- Beschreibungen/Notizen
- Abstract Candida albicans is a major cause of opportunistic and life-threatening systemic fungal infections, especially in the immunocompromised. The plasma membrane proton-pumping ATPase (Pma1p) is an essential enzyme that generates the electrochemical gradient required for cell growth. We expressed C. albicans Pma1p (CaPma1p) in Saccharomyces cerevisiae to facilitate screening for inhibitors. Replacement of S. cerevisiae PMA1 with C. albicans PMA1 gave clones expressing CaPma1p that grew slowly at low pH. CaPma1p was expressed at significantly lower levels and had lower specific activity than the native Pma1p. It also conferred pH sensitivity, hygromycin B resistance, and low levels of glucose-dependent proton pumping. Recombination between CaPMA1 and the homologous nonessential ScPMA2 resulted in chimeric suppressor mutants that expressed functional CaPma1p with improved H+-ATPase activity and growth rates at low pH. Molecular models of suppressor mutants identified specific amino acids (between 531 and 595 in CaPma1p) that may affect regulation of the activity of Pma1p oligomers in S. cerevisiae. A modified CaPma1p chimeric construct containing only 5 amino acids from ScPma2p enabled the expression of a fully functional enzyme for drug screens and structural resolution.
- Sprache
- Englisch
- Identifikatoren
- ISSN: 1567-1356eISSN: 1567-1364DOI: 10.1111/1567-1364.12035Titel-ID: cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_4428687
- Format
- –
- Schlagworte
- Adenosine triphosphatase, Amino acids, antifungal drug target, Candida albicans, Candida albicans - enzymology, Candida albicans - genetics, Culture Media - chemistry, Drug Evaluation, Preclinical - methods, Drug screening, Enzyme Inhibitors - isolation & purification, Enzymes, Gene Expression, Growth rate, H+-transporting ATPase, Hydrogen-Ion Concentration, Hygromycin, Hygromycin B, Models, Molecular, Molecular modelling, Mutants, pH effects, plasma membrane H+‐ATPase, PMA1, Protein Conformation, proton pump, Proton-Translocating ATPases - chemistry, Proton-Translocating ATPases - genetics, Proton-Translocating ATPases - metabolism, Recombinant Proteins - chemistry, Recombinant Proteins - genetics, Recombinant Proteins - metabolism, Recombination, Recombination, Genetic, Saccharomyces cerevisiae, Saccharomyces cerevisiae - enzymology, Saccharomyces cerevisiae - genetics, Saccharomyces cerevisiae - growth & development, Suppression, Genetic, Yeast