Two different genes from Schwanniomyces occidentalis determine ribosomal resistance to cycloheximide

Abstract

Two genes (SCRI and SCR2) encoding natural cycloheximide resistance in the budding yeast Schwanniomyces occidentalis have been cloned by expression in Saccharomyces cerevisiae. Both genes determine resistance to the inhibitory action of cycloheximide on the ribosome. SCRl and SCR2 are present as single copies in Schwanniomyces occidentalis, where they map on chromosomes I1 and V, respectively. The nucleotide sequence of SCR2 contains an open reading frame of 321 nucleotides which is interrupted by an intron of 452 nucleotides. It encodes a polypeptide of 106 amino acids of molecular mass 12.25 kDa and PI 11.19. The deduced amino acid sequence shows a high degree of similarity to the LA1 protein of the 60s ribosomal subunit from several eukaryotic organisms. The intron and the 5' non-coding region of SCR2 possess conserved elements which are typical of yeast ribosomal protein genes. A single amino acid change determines the resistance or sensitive phenotype to cycloheximide of the 80s ribosome since replacement of Gln56 in LA1 from Schwanniomyces with Pro, by site-directed mutagenesis, confers cycloheximide sensitivity. SCR2 may serve as a practical yeast cloning marker if integrated in a multicopy plasmid. [l] is a budding yeast which has attracted the attention of researchers because it secrets a variety of high-molecular-mass proteins. These include a-amylase and a glucoamylase which also shares a starch debranching activity. Therefore it solubilizes starch by complete hydrolysis to glucose [2]. Because of the potential industrial importance of this yeast, it is desirable to characterize in detail its genetic constitution and its spectrum of sensitivity or resistance to antibiotics. Indeed Sw. occidentalis is highly resistant in vivo to inhibition by the glutarimide antibiotic cycloheximide (cyh) [3], which blocks polypeptide chain elongation by eukaryotic ribosomes (for reviews, see [4,51). This resistance is also shared by certain other yeast species including Kluyveromyces lactis and K. fragilis [6, 71, Candida maltosa and C. tropicalis [8, 91. In Saccharomyces cerevisiae, mutations at five different loci induce resistance to cyh [lo, 111. Of these, only cyh2 has been subjected to detailed biochemical and genetic studies. The presence of this gene confers resistance to cyh levels of 50-70 pg/ml. The gene encodes an altered ribosomal protein L29 which is presumed to reduce the affinity of the 60s ribosomal subunit for cyh [12, 131. The cyh2 gene has been Correspondence to A. Schwanniomyces occidentalis

The SCR1 gene from Schwanniomyces occidentalis encodes a highly hydrophobic polypeptide, which confers ribosomal resistance to cycloheximide

“…YEp356r plasmid has been described previously (Myers et al, 1986). The multicopy YEp13 and monocopy YCp50 S. cerevisiae-E. coli shuttle vectors, as well as pSC7 and pSC2.1 plasmids, containing the Sw. occidentalis SCR1 gene, have been described elsewhere (del Pozo et al, 1993). Plasmids pSCR1 and pYCp2.1, containing the SCR1 gene, were constructed by cloning a 3.8 kb EcoRI-SalI fragment from pSC2.1 in EcoRI-SalIdigested pBS and YCp50 plasmids, respectively ( Figure 1).…”

“…The nucleotide sequence of SCR2 encodes a polypeptide of 106 amino acids that shows a high similarity to those of the L42 protein of the 60S ribosomal subunit from several eukaryotic organisms. A single amino acid (Gln56) in L42 from Sw. occidentalis was shown to determine cyh resistant 80S ribosome in S. cerevisiae (del Pozo et al, 1993).…”

“…Numerous yeasts, including Saccharomyces cerevisiae, are highly sensitive to low concentrations (approximately 0.5 mg/ml) of this antibiotic. In contrast, other yeasts, including Schwanniomyces occidentalis (Debaromyces occidentalis; Barnett et al, 1983;del Pozo et al, 1993), Kluyveromyces lactis, K. fragilis (Adoutte-Panvier and Davies, 1984;Rao and Grollman, 1967), Candida maltosa, C. tropicalis (Takagi et al, 1985(Takagi et al, , 1986, Phaffia rhodozyma (Kim et al, 1998) and Cryptococcus neoformants (Varma and Kwon-Chung, 2000) are naturally resistant to cyh at rather high concentrations (i100 mg/ml). Resistance of cyh in yeast cells occurs by either modification of ribosomal proteins or expression of certain specific transport systems responsible for a multidrug resistance generally linked to the expression of MDR genes.…”

“…With respect to the first case, a single amino acid change at ribosomal proteins (rps) L29 or L42 (formerly L41; Mager et al, 1997) promotes the cyh sensitivity (cyh S )/resistance (cyh r ) property of the 80S ribosome from C. maltosa (Takagi et al, 1985(Takagi et al, , 1986Mutoh et al, 1995), Cr. neoformans (Varma and Kwon-Chung, 2000) Sw. occidentalis (del Pozo et al, 1993) or P. rhodozyma (Kim et al, 1998). In relation to the second resistance mechanism, numerous yeast MDR genes have been described.…”

“…occidentalis presents high constitutive cyh r levels and grows normally at >1000 mg/ml drug concentrations (Barnett et al, 1983). Previously, we reported that two genes (SCR1 and SCR2) from the budding yeast Sw. occidentalis were cloned and expressed in S. cerevisiae (del Pozo et al, 1993). Both genes determine cyh-resistant ribosomes.…”