title: Heterologous expression of Candida albicans Pma1p in Saccharomyces cerevisiae.
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.
authors: The Sir John Walsh Research Institute, School of Dentistry, University of Otago, Dunedin, New Zealand. mikhail.keniya@otago.ac.nz
authors: Mikhail V
authors: MV
authors: Keniya
authors: Richard D
authors: RD
authors: Cannon
authors: ÂnBình
authors: Â
authors: Nguyễn
authors: Joel D A
authors: JD
authors: Tyndall
authors: Brian C
authors: BC
authors: Monk
mesh_terms: D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004791:Enzyme Inhibitors / Q000302:isolation & purification; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D013489:Suppression, Genetic
chemical_list: D003470:Culture Media; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases
references: Biochim Biophys Acta. 1996 Oct 23;1284(2):181-90
references: 8914582
references: Ann N Y Acad Sci. 1997 Nov 3;834:609-17
references: 9405872
references: Nature. 1986 Feb 20-26;319(6055):689-93
references: 3005867
references: Proc Natl Acad Sci U S A. 2011 Feb 1;108(5):1833-8
references: 21239683
references: Biochim Biophys Acta. 2009 Apr;1787(4):207-20
references: 19388138
references: Biochim Biophys Acta. 1998 Jul 17;1372(2):261-71
references: 9675306
references: J Enzyme Inhib Med Chem. 2010 Jun;25(3):312-7
references: 20210698
references: Biochim Biophys Acta. 1999 Feb 4;1417(1):32-6
references: 10076033
references: J Biol Chem. 2007 Dec 7;282(49):35471-81
references: 17932035
references: J Biol Chem. 1994 Apr 8;269(14):10393-9
references: 8144622
references: J Biol Chem. 1995 Oct 6;270(40):23828-37
references: 7559560
references: Eukaryot Cell. 2007 Jul;6(7):1150-65
references: 17513564
references: Methods Enzymol. 1988;157:528-33
references: 2906716
references: Crit Rev Microbiol. 1994;20(3):209-23
references: 7802957
references: Curr Protoc Bioinformatics. 2006 Oct;Chapter 5:Unit-5.6
references: 18428767
references: Nucleic Acids Res. 2003 Jul 1;31(13):3503-6
references: 12824354
references: J Biol Chem. 1991 Sep 25;266(27):18097-103
references: 1833392
references: Gene. 2005 Nov 21;361:57-66
references: 16185821
references: Nature. 1970 Aug 15;227(5259):680-5
references: 5432063
references: J Biol Chem. 1993 Sep 15;268(26):19744-52
references: 8366114
references: Clin Microbiol Rev. 2007 Jan;20(1):133-63
references: 17223626
references: Nature. 1998 Apr 23;392(6678):840-3
references: 9572146
references: Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10531-5
references: 7937988
references: J Cell Biol. 1991 Oct;115(2):289-95
references: 1833410
references: Mol Biol Cell. 2001 Dec;12(12):4129-38
references: 11739806
references: Nucleic Acids Res. 1996 Jul 1;24(13):2519-24
references: 8692690
references: Nature. 2007 Dec 13;450(7172):1111-4
references: 18075595
references: FEBS Lett. 1987 Nov 16;224(1):187-92
references: 2960558
references: J Bacteriol. 1991 Nov;173(21):6826-36
references: 1834633
references: J Biol Chem. 1998 May 15;273(20):12612-22
references: 9575223
references: J Biol Chem. 2006 Aug 18;281(33):23887-98
references: 16751629
references: J Biol Chem. 1994 Sep 30;269(39):23988-95
references: 7929048
references: Antimicrob Agents Chemother. 2005 Jan;49(1):57-70
references: 15616276
references: Acta Biochim Pol. 1995;42(4):481-96
references: 8852338
references: Anal Biochem. 1981 Jan 1;110(1):201-7
references: 6163373
references: Arch Biochem Biophys. 2008 Aug 1;476(1):3-11
references: 18455499