Purification of geranylgeranyltransferase I from Candida albicans and cloning of the CaRAM2 and CaCDC43 genes encoding its subunits.
All previously characterized protein geranylgeranyltransferases I (GGTase I) are heterodimeric zinc metalloenzymes which catalyse geranylgeranylation of a cysteine residue in proteins containing a C-terminal CaaL motif (C, Cys; a, aliphatic amino acid; L, Leu). The alpha and beta subunits of GGTase I of Saccharomyces cerevisiae are encoded by RAM2 and CDC43, respectively, and are essential for yeast viability. The authors are therefore investigating the role of geranylgeranylation in the related pathogenic yeast, Candida albicans, which is the most prevalent human fungal pathogen. GGTase I was purified to near homogeneity and also found to be a heterodimeric magnesium-dependent, zinc metalloenzyme displaying selectivity for CaaL-containing protein substrates. GGTase I peptide sequences were obtained from the purified protein and used to clone the genes encoding both subunits. CaRAM2 and CaCDC43 encode proteins that are 42 and 34% identical to their corresponding S. cerevisiae homologues, respectively, and 30% identical to their human homologues. Despite the limited overall homology, key zinc- and substrate-binding residues of the beta subunit (Cdc43p) are conserved. A unique feature of CaCdc43p is a tract of polyasparagine whose length varies from 6 to 17 residues among C. albicans strains and between alleles. Coexpression of both CaCDC43 and CaRAM2 under their native promoters complemented the ts defect of a S. cerevisiae cdc43 mutant but expression of the beta-subunit alone did not correct the growth defect, suggesting that hybrid GGTase I heterodimers are nonfunctional.
Mazur
Paul
P
Register
Elizabeth
E
Bonfiglio
Cynthia A
CA
Yuan
Xiling
X
Kurtz
Myra B
MB
Williamson
Joanne M
JM
Kelly
Rosemarie
R
D019883:Alkyl and Aryl Transferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002176:Candida albicans / Q000201:enzymology* / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D017368:Protein Prenylation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D014166:Transferases
D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; C077378:RAM2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; C065203:CDC43 protein, S cerevisiae