Ribosomal P-protein Stalk Function Is Targeted by Sordarin Antifungals

Marı́A G Gómez-Lorenzo,José F Garcı́A-Bustos

doi:10.1074/jbc.273.39.25041

Abstract

Sordarin derivatives are remarkably selective inhibitors of fungal protein synthesis. Available evidence points to a binding site for these inhibitors on elongation factor 2, but high affinity binding requires the presence of ribosomes. The gene mutated in one of the two isolated complementation groups of Saccharomyces cerevisiae mutants resistant to the sordarin derivative GM193663 has now been identified. It is RPP0, encoding the essential protein of the large ribosomal subunit stalk rpP0. Resistant mutants are found to retain most of the binding capacity for the drug, indicating that mutations in rpP0 endow the ribosome with the capacity to perform translation elongation in the presence of the inhibitor. Other proteins of the ribosomal stalk influence the expression of resistance, pointing to a wealth of interactions between stalk components and elongation factors. The involvement of multiple elements of the translation machinery in the mode of action of sordarin antifungals may explain the large selectivity of these compounds, even though the individual target components are highly conserved proteins.

Highlights

The ribosomal stalk is a defined morphological feature most evident in prokaryotic ribosomes, where it is composed of the L7/L12 tetramer, L10 and L11 proteins, and a highly conserved domain of the 23 S rRNA termed the “GTPase center.” The stalk plays a prominent role in the elongation phase of ribosomal protein synthesis
Sordarin inhibitors seem to bind to elongation factor 21,2 [18], and point mutations on the factor make cells resistant to the inhibitors1 [18], but high affinity binding requires the presence of ribosomes,2 and resistant mutants were detected, which did not map on EF2.1
All mutants displayed cross-resistance on plates to other members of the sordarin family, but not to the protein synthesis inhibitors cycloheximide, hygromycin, or verrucarin A, showing that sordarins act by a mechanism different from the one used by those other inhibitors

Summary

EXPERIMENTAL PROCEDURES

Strains and Growth Conditions—All the strains used in this study are derivatives of: S. cerevisiae SEY6210 (Mat ␣ ura leu 112 his3⌬200 trp1⌬901 lys suc2⌬9), its ade101 derivative SEY6211 Jimenez), S. cerevisiae W303 (Mat a leu 112 trp his 3–11, 15 ade 2-1 can 1–100) and S. cerevisiae W303dGP0 (W303 strain with the cassette pGAL1-rpP0-URA3 replacing the endogenous RPP0 locus) [19]. For genetic mapping of the FPR2–15 mutation, crosses were made between strain W303dGP0 and W303 derivatives D46 (rpp1b::TRP1 rpp2a::URA3) and D57 (rpp1a::LEU2 rpp2b::HIS3). Heterologous rpP0 proteins were expressed in the W303dGP0 strain growing in glucose, to repress the chromosomal RPP0 gene, using the centromeric plasmid pFL39-HIS and the endogenous S. cerevisiae promoter. Spontaneous resistant mutants were selected on YPD medium plates with 100 ␮g/ml sordarin derivative GM193663.1 P-protein gene disruptions on strains 6210, FPR1–19, and FPR2–15 were carried out according to standard procedures by allele replacement, using different constructions for genes rpp1a, rpp1b, rpp2a, and rpp2b, all obtained from the laboratory of J. Chemother., in press. 3 The abbreviation used is: PCR(s), polymerase chain reaction(s)

Ribosomal Stalk Proteins and Sordarin Antifungals

RESULTS

TABLE II

DISCUSSION