Open AccessHeterologous expression of C andida albicans Pma1p in S accharomyces cerevisiae
Author(s) -
Keniya Mikhail V.,
Can Richard D.,
Nguyễn ẤnBình,
Tyndall Joel D.A.,
Monk Brian C.
Publication year - 2013
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/1567-1364.12035
Subject(s) - biology , candida albicans , saccharomyces cerevisiae , hygromycin b , biochemistry , mutant , corpus albicans , heterologous expression , enzyme , amino acid , heterologous , microbiology and biotechnology , yeast , recombinant dna , gene
C andida 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 S accharomyces 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.