Purification of geranylgeranyltransferase I from Candida albicans and cloning of the CaRAM2 and CaCDC43 genes encoding its subunits

Abstract

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 a and /?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 magnesiumdependent, 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 5. cerevisiae homologues, respectively, and 30 O/ O identical to their human homologues. Despite the limited overall homology, key zinc-and substrate-binding residues of the /? 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 /?-subunit alone did not correct the growth defect, suggesting that hybrid GGTase I heterodimers are nonfunctional.

Geranylgeranyltransferase I of Candida albicans : Null Mutants or Enzyme Inhibitors Produce Unexpected Phenotypes

“…Rho1p and Cdc42p of C. albicans contain CaaL motifs and are likely GGTase I substrates in vivo (22,28). Previously, we reported the purification of C. albicans GGTase I and the cloning and sequence analysis of its ␣ and ␤ subunit genes (27). Our work showed that C. albicans GGTase I is also a zinc-dependent, magnesium-dependent heterodimer whose subunits demonstrated 30% amino acid identity with their human counterparts.…”

“…Protein PTase activities were determined by an acid quench-filtration assay as previously described (27) All reactions were performed in duplicate and were initiated by the addition of PTase activity (2.5 to 5 l) to a final volume of 25 l and incubated at 30°C for 30 to 60 min. All other details of the assay and the origin of the Ras substrates were described previously (27).…”

“…The probe was a radiolabeled 2.7-kb CaCDC43 HindIII fragment. tion of this acceptor (27). To confirm this point, GGTase I and FTase activities were partially resolved by anion-exchange chromatography, and column fractions were assayed for PTase activity with Ras-CVIL and GGPP and with Ras-CVLM and FPP.…”

“…We previously showed, using partially purified PTases, that C. albicans GGTase I demonstrated a strong preference for Ras-CaaX substrates in which X of the CaaX motif was a leucine, as was true for both the Saccharomyces and mammalian GGTase I (27). C. albicans FTase demonstrated strong activity with Ras-CVLM, as does S. cerevisiae FTase, and also farnesylated all Ras-CaaL substrates tested at levels ranging from 2 to 20% relative to that observed with Ras-CVLM.…”

Structure of Protein Geranylgeranyltransferase-I from the Human Pathogen Candida albicans Complexed with a Lipid Substrate

“…In pathogenic microorganisms, such as C. albicans, disruption of protein prenylation of such essential cellular proteins has the potential for the development of new antifungal medications (5)(6)(7)(8)23). The Ram2 gene in C. albicans encodes the common ␣-subunit of FTase and GGTase-I; knock-out mutations of this gene are lethal (5).…”