Temperature-sensitive mutation in yeast mitochondrial ribosome recycling factor (RRF)

Abstract

The yeast protein Rrf1p encoded by the FIL1 nuclear gene bears significant sequence similarity to Escherichia coli ribosome recycling factor (RRF). Here, we call FIL1 Ribosome Recycling Factor of yeast, RRF1. Its gene product, Rrf1p, was localized in mitochondria. Deletion of RRF1 leads to a respiratory incompetent phenotype and to instability of the mitochondrial genome (conversion to rho(-)/rho(0) cytoplasmic petites). Yeast with intact mitochondria and with deleted genomic RRF1 that harbors a plasmid carrying RRF1 was prepared from spores of heterozygous diploid yeast. Such yeast with a mutated allele of RRF1, rrf1-L209P, grew on a non-fermentable carbon source at 30 but not at 36 degrees C, where mitochondrial but not total protein synthesis was 90% inhibited. We propose that Rrf1p is essential for mitochondrial protein synthesis and acts as a RRF in mitochondria.

Figure 1

Haploid yeast with a RRF1 deletion does not grow on glycerol, a phenotype that cannot be overcome by cytoplasmic Rrf1p. (A) Immunodetection of Rrf1p in Δrrf1 haploid cells (WY347) transformed with a centromeric copy of the RRF1 gene. Total protein extracts from four different strains were analyzed, shown from left to right: wild-type RRF1 haploid cells (WY344, designated RRF1 cells) harboring a centromeric control plasmid carrying no RRF1 (pRS415-ADHp); WY344 harboring a centromeric plasmid carrying the RRF1 gene (designated pRRF1W1); Δrrf1 haploid cells (WY347, designated Δrrf1 cells) harboring pRS415-ADHp or harboring pRRF1W1. Protein extracts (50 µg) were analyzed in 12% gels by western blotting using an antibody against His₆-partial Rrf1p. (B) Lack of growth of Δrrf1 haploid cells harboring a plasmid carrying RRF1 on a non-fermentable carbon source. Four different strains were streaked on an agar plate containing ethanol selective medium. Wild-type RRF1 haploid cells (WY344, designated RRF1) harboring a centromeric control plasmid carrying no RRF1 (pRS415-ADHp) (top right). WY344, harboring a centromeric plasmid carrying the RRF1 gene (pRRF1W1) (top left). Δrrf1 haploid cells (WY347, designated Δrrf1) harboring pRS415-ADHp (bottom right). WY347 harboring pRRF1W1 (bottom left). The photograph was taken after 3 days growth at 30°C.

Figure 2

Complementation of Δrrf1 rho⁺ haploid cells with a plasmid carrying RRF1. Evidence that rrf1-L209P gives temperature-sensitive growth. Δrrf1 rho⁺ haploid cells harboring various plasmids carrying mutated RRF1 were created as described in Materials and Methods. These various strains are indicated in the circle with the pie cut (left). (A) Cells harboring each of the plasmids as shown in the left panel were streaked onto synthetic glucose (3%) medium lacking leucine and grown at 24, 30 and 36°C. (B) The same strains were grown overnight in synthetic glucose medium minus leucine, streaked onto YPG plates (containing 3% glycerol) and incubated at 24, 30 and 36°C. Δrrf1 rho⁺ haploid cells (ET2) are ade2-101. Such adenine-deficient cells are known to accumulate red pigments in the presence of low adenine when they are respiratory competent. Note that Δrrf1 rho⁺ + prrf-L209P could not grow on glycerol at 36°C (lower middle streak, plate 3) while the strain harboring pRS415-ADHp (no RRF1) did not grow in glycerol medium at all temperatures tested (plates 1–3, upper right streak). This indicates that Δrrf1 rho⁺ + pRS415-ADHp was converted to rho^– because of the lack of Rrf1p.

Figure 3

(I) Rrf1p is localized in the fraction containing mitochondria. Western blot analysis of crude yeast extracts (designated column C), isolated mitochondria (column M) and post-mitochondrial supernatant fraction (C-M) obtained from wild-type strain DS413. Proteins (40 µg) of each preparation were analyzed by western blotting using antibody against His₆-partial Rrf1p. (II) Cytological localization of Rrf1p to mitochondria. A representative microscopic field of Δrrf1 rho⁺ haploid cells (strain ET2) harboring a high copy number plasmid carrying RRF1 (pRRF1W2) is shown. (A) Differential interference contrast view. (B) DAPI staining showing the locations of nuclear and mitochondrial DNA. (C) Indirect immunofluorescence using antibodies to His₆-partial Rrf1p and FITC- conjugated secondary antibody. Bar represents 1 µm.

Figure 4

Cells with mutant Rrf1p (L209P) synthesize much less mitochondrial protein than those with wild-type Rrf1p. Haploid Δrrf/rho⁺ cells expressing wild-type Rrf1p (ET2 + pRRF1W1) and thermosensitive Rrf1p (ET2 + prrf1-L209P) were grown overnight at 30°C in minimal glucose medium lacking methionine until mid-exponential phase. Cells were then labeled with [³⁵S]methionine for 80 min in the presence of cycloheximide at 37°C. Whole cell extracts (∼0.5 OD₆₀₀ units) were separated on a 12% polyacrylamide gel. The gel was Coomassie stained, dried and exposed to X-ray film. (A) and (B) are Coomassie staining and autoradiography, respectively. The mitochondrial proteins revealed by autoradiography were identified as Cox1p, Cox2p, Cox3p, Cobp (cytochrome b), Atp6p and Oli1p (Atp9p) (subunits 6 and 9 of the F₀F₁-ATPase, respectively) and Var1p (variant ribosomal protein). The positions of the molecular weight markers are indicated on the left (in kDa).

Figure 5

Incorporation of [³⁵S]methionine into total proteins by haploid cells expressing thermosensitive Rrf1p at a permissive (29°C) and a non-permissive temperature (37°C) are similar. Translation activities of haploid Δrrf1 rho⁺ cells expressing wild-type Rrf1p (ET2 + pRRF1W1, left panel) and thermosensitive Rrf1p (ET2 + prrf1-L209P, right panel) were examined. Overnight cultures in glucose were diluted to 0.05 OD₅₄₀ and [³⁵S]methionine was added. Cells were incubated at 29°C until the OD₅₄₀ reached 0.45. Then (indicated by arrow), one sample was exposed to 37°C (closed circles or triangles and dot line) while the other was kept at 29°C (open circles or triangles and solid line). The incorporation of radioactive methionine into the hot TCA-insoluble fraction after the temperature shift is shown.

Figure 6

Predicted surface of Rrf1p. A Connolly surface was calculated as described in Materials and Methods for the Rrf1p protein using a theoretical sphere of 2.4 Å. The green color indicates the portion corresponding to the D loop of tRNA.