Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts

Abstract

Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.

Expression of a Micro-protein

“…Thermus thermophilus (10). (BEWARE: the name L41 has also been applied to a different fungal ribosomal protein, mutant alleles of which can cause resistance to cycloheximide (11,12). In the official nomenclature that protein is now known as L42 (13).…”

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

“…In contrast, ribosomal-resistance to cyh in S. cerevisiae is conferred by a single amino acid change at rp L29, which is encoded in the cyh2 gene (Stö cklein and Piepersberg, 1980). A similar situation is found in Sw. occidentalis and Kluyveromyces lactis, where a single amino acid change occurs at a rp equivalent to L42 from S. cerevisiae (Kawai et al, 1992;del Dehoux et al, 1993). In all these cases, resistance is thought to involve a conformational change of the 60S ribosomal subunit, which reduces its affinity for cyh.…”

Construction and Some Characterization of a Yeast Artificial Chromosome Library from DNA of a Tomato Line Having Four Disease Resistance Traits

“…3A) suggesting that the tomato has two kinds of LEL41 genes as is the case of Saccharomyces cerevisiae. 19 ) Colonies of the two batches (batch 11 and batch 12) were then hybridized separately with the radiolabeled LEL41 cDNA and a single positive clone was identified in batch 12 (Fig. 3C).…”