The RAM1 gene encoding a protein- farnesyltransferase b-subunit homologue is essential in Cryptococcus neoformans Marcelo A. Vallim, Larissa Fernandes3 and J. Andrew Alspaugh Correspondence J. Andrew Alspaugh andrew.alspaugh@duke.edu Department of Medicine and Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA Received 13 January 2004 Revised 9 March 2004 Accepted 12 March 2004 Many small G proteins require post-translational modification to allow functional association to the cell membrane. This process often involves the enzymic addition of hydrophobic prenyl groups to a conserved cysteine residue near the C-terminus of the protein. The enzymes that catalyse these reactions include protein farnesyltransferase and protein geranylgeranyltransferases. The human fungal pathogen Cryptococcus neoformans requires functional Ras and Rho proteins in order to undergo normal growth and differentiation. Since farnesylation and geranylgeranylation are likely required for the proper function of these small G proteins, we hypothesized that inhibition of these prenylation events would alter the growth and cellular morphogenesis of this fungus. We cloned the RAM1 gene encoding the single protein-farnesyltransferase b-chain homologue in C. neoformans. Using a gene-disruption strategy in a diploid C. neoformans strain, we demonstrated that this gene encodes an essential function, in contrast to the case in Saccharomyces cerevisiae, in which the homologous RAM1 gene is not essential for growth. Pharmacological inhibition of farnesyltransferase activity resulted in dose-dependent cytostasis of C. neoformans, as well as prevention of hyphal differentiation. Simultaneous inhibition of farnesylation and calcineurin signalling results in a synthetic effect on growth. Protein farnesylation is required for the growth and cellular differentiation of C. neoformans and may provide novel targets for antifungal therapy. INTRODUCTION Cryptococcus neoformans is an opportunistic human fungal pathogen. Several small G proteins have previously been demonstrated to regulate the growth and development of this fungus. For example, genetic studies demonstrated that the RHO1 gene is likely essential, as it is in other fungal species, and that precise regulation of the Rho1 protein is required for sustained growth of C. neoformans (Chang et al., 2000). Additionally, the small G protein Ras1 is required for proper growth, development and pathogenicity of C. neoformans. A null mutation of the RAS1 gene results in a mutant strain unable to grow at 37 uC, rendering it avirulent in animal models of cryptococcal disease. Ras1 is also required for hyphal formation of this fungus; ras1- mutant strains are defective in mating and haploid fruiting, two types of differentiation characterized by a yeast–hyphal transition (Alspaugh et al., 2000). In order for small G proteins such as Ras and Rho to localize to the cell membrane and to function properly, they must undergo prenylation, a post-translational modification in which hydrophobic groups are added to the C-terminus of the protein (Casey et al., 1996). Three prenyltransferases have been described in eukaryotic cells that are responsible for these protein modifications: protein farnesyltransferase (FTase) and protein geranylgeranyltransferase (GGTase) types I and II. FTase catalyses the addition of 15-carbon (farnesyl) groups to proteins destined for cell membranes (Reiss et al., 1990), and GGTases catalyse a similar reac- tion with 20-carbon (geranylgeranyl) groups (Seabra et al., 1991). Since activating Ras mutations have been demon- strated in many human malignancies (Schafer et al., 1989), and since prenylation is required for Ras function (Casey et al., 1989; Hancock et al., 1989), these prenyltransferases have been extensively studied as potential targets for cancer chemotherapy (Hill et al., 2000; Lerner et al., 1995; Prendergast, 2000). FTase and GGTase I are related heterodimeric proteins that consist of a common a subunit and distinct b subunits. In Saccharomyces cerevisiae, the RAM2 gene encodes the common a subunit of FTase and GGTase I, and the RAM1 and CDC43 genes encode the b subunits of FTase and 3Present address: Departamento de Biologia Celular, IB, UnB, Brasilia, DF. The GenBank accession number for the C. neoformans RAM1 gene sequence reported in this paper is AY162319. Abbreviations: FTase, protein farnesyltransferase; GGTase, protein geranylgeranyltransferase; RACE, Rapid Amplification of cDNA Ends. 0002-7030 G 2004 SGM Printed in Great Britain 1925 Microbiology (2004), 150, 1925–1935 DOI 10.1099/mic.0.27030-0