SYM1 is the stress-induced Saccharomyces cerevisiae ortholog of the mammalian kidney disease gene Mpv17 and is required for ethanol metabolism and tolerance during heat shock

Abstract

Organisms rapidly adapt to severe environmental stress by inducing the expression of a wide array of heat shock proteins as part of a larger cellular response program. We have used a genomics approach to identify novel heat shock-induced genes in Saccharomyces cerevisiae. The uncharacterized open reading frame (ORF) YLR251W was found to be required for both metabolism and tolerance of ethanol during heat shock. YLR251W has significant homology to the mammalian peroxisomal membrane protein Mpv17, and Mpv17(-/-) mice exhibit age-onset glomerulosclerosis, deafness, hypertension, and, ultimately, death by renal failure. Expression of Mpv17 in ylr251wdelta cells complements the 37 degrees C ethanol growth defect, suggesting that these proteins are functional orthologs. We have therefore renamed ORF YLR251W as SYM1 (for "stress-inducible yeast Mpv17"). In contrast to the peroxisomal localization of Mpv17, we find that Sym1 is an integral membrane protein of the inner mitochondrial membrane. In addition, transcriptional profiling of sym1delta cells uncovered changes in gene expression, including dysregulation of a number of ethanol-repressed genes, exclusively at 37 degrees C relative to wild-type results. Together, these data suggest an important metabolic role for Sym1 in mitochondrial function during heat shock. Furthermore, this study establishes Sym1 as a potential model for understanding the role of Mpv17 in kidney disease and cardiovascular biology.

FIG. 1.

sym1Δ mutant cells are hypersensitive to ethanol and heat shock. (A) Wild-type and sym1Δ strains were plated on SC medium containing glucose (2%), ethanol (2%), glycerol (3%), or glycerol plus ethanol and incubated at 30 or 37°C. Plates with nonfermentable carbon sources were incubated for several days longer than glucose plates due to slower growth rates. (B) Wild-type and sym1Δ strains were plated on SC medium containing glycerol (3%) plus the indicated concentrations of ethanol and grown at 30 and 37°C for the same length of time. Because no growth effects were observed at 30°C, only the 37°C series is shown.

FIG. 2.

SYM1 is stress inducible. (A) A wild-type culture in mid-logarithmic-growth phase was subjected to a 1-h heat shock at 37°C, aliquots were removed at the indicated time points, and total RNA was prepared for Northern analysis as described in Materials and Methods. Radiolabeled probes were used to detect mRNA levels of the indicated genes. STI1 is a well-established heat shock gene, and PGK1 is used as a loading control. (B) Sym1 protein levels after a brief heat shock were examined in the sym1Δ mutant strain transformed with a plasmid expressing SYM1 tagged with a triple-HA epitope at the carboxyl terminus under the control of its own promoter. Equal amounts of mid-logarithmic-phase cells were harvested at 30°C or after 15 min of heat shock at 37°C, protein extracts were prepared, and the levels of Sym1-HA and the loading control PGK were analyzed by Western blotting. (C) Protein extracts were prepared from the same strain used as described for panel B from cells grown in glucose medium or after a 9-h shift to ethanol-containing medium and were analyzed by Western blotting. The polyclonal antibody used to detect the mitochondrial ATP/ADP carrier protein (AAC) also recognizes the mitochondrial outer membrane protein porin.

FIG. 3.

sym1Δ cells show reversible cessation of growth on ethanol during heat shock and differential sensitivity to ethanol catabolic intermediates. (A) Wild-type and sym1Δ cells were grown in YPD medium at 30°C, diluted into YP-2% ethanol medium, and grown to mid-log phase and then rediluted to an optical density at 600 nm (OD₆₀₀) of 0.1 in YP-2% ethanol, and growth was monitored at 37°C for 28 h. (B) Dilutions of wild-type and sym1Δ cells were spotted onto SC medium containing 2% ethanol and grown for 7 days at 37°C. The same plates were then shifted to 30°C for an additional 3 days. (C) Dilutions of wild-type and sym1Δ cells were spotted onto SC medium containing ethanol (2%), acetaldehyde (0.1%), or acetate (2%) and grown at both 30 and 37°C. Incubation times differed for each carbon source but were identical for both strains. Because no growth defects were observed at 30°C, only the 37°C plates are shown. Note the smaller colony size of the sym1Δ strain and acetaldehyde and the absence of a defect on acetate.

FIG. 4.

SYM1 is the yeast functional ortholog of the kidney disease gene Mpv17. (A) CLUSTAL alignment of the protein coding sequences of SYM1, Homo sapiens Mpv17, Mus musculus Mpv17, and M. musculus Pmp22. Identical residues are boxed. The four predicted transmembrane domains (TM1, TM2, TM3, and TM4) are indicated with bars over the protein sequences. (B) Hydropathy profiles of the SYM1 and Mpv17 protein coding sequences were generated using TMPred. Putative transmembrane domains are indicated. (C) Dilutions of wild-type and sym1Δ cells carrying either pRS426GPD (vector) or pRS426GPDHsMPV17 were spotted onto SC-URA plates containing 2% ethanol as sole carbon source and incubated at 37°C.

FIG. 5.

Sym1 is localized to the mitochondrial network. (A) Wild-type or sym1Δ cells carrying pRS416GPD (vector) or pRS416GPDSym1-GFP were spotted onto SC-URA plates containing 2% ethanol as sole carbon source and incubated at 37°C. (B) sym1Δ cells carrying either pRS416GPDSym1-GFP or pRS416GPDCFP-skl were grown overnight in selective medium and stained with the mitochondrial dye MitoTracker Red. Cells were photographed under epifluorescence illumination with the appropriate filters to detect the fluorescent protein fusions (FP) and MitoTracker signal (MT) or using differential interference contrast optics (DIC). (C) Wild-type, pex3Δ, and pex19Δ cells transformed with either pRS416GPDSym1-GFP or pRS413GPDCFP-skl were grown overnight in selective medium and photographed as described above.

FIG. 5.

Sym1 is localized to the mitochondrial network. (A) Wild-type or sym1Δ cells carrying pRS416GPD (vector) or pRS416GPDSym1-GFP were spotted onto SC-URA plates containing 2% ethanol as sole carbon source and incubated at 37°C. (B) sym1Δ cells carrying either pRS416GPDSym1-GFP or pRS416GPDCFP-skl were grown overnight in selective medium and stained with the mitochondrial dye MitoTracker Red. Cells were photographed under epifluorescence illumination with the appropriate filters to detect the fluorescent protein fusions (FP) and MitoTracker signal (MT) or using differential interference contrast optics (DIC). (C) Wild-type, pex3Δ, and pex19Δ cells transformed with either pRS416GPDSym1-GFP or pRS413GPDCFP-skl were grown overnight in selective medium and photographed as described above.

FIG. 6.

Sym1 is an integral membrane protein of the mitochondrial inner membrane. (A) sym1Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, spheroplasted, and lysed by Dounce homogenization. The sample was split into two equal aliquots, one of which was held as the total cell extract (T); the other was subjected to centrifugation at 16,100 × g, and both supernatant (S) and membrane pellet (P) fractions were isolated. Equivalent amounts of all fractions were subjected to SDS-PAGE and Western blotting to identify the indicated proteins. (B) sym1Δ cells expressing the Sym1-HA fusion were grown in SC-URA, harvested, and lysed using glass beads. A medium-speed membrane pellet was generated by centrifugation at 16,100 × g. The membrane pellet was split into thirds and treated with buffer, 0.1 M NaCO₃, or 1% Triton X-100, and the aliquots were again centrifuged at 16,100 × g to isolate membrane pellet (P) and supernatant (S) fractions. SDS-PAGE and Western blotting was carried out as described for panel A. (C) Mitochondria were isolated for the protease protection assay as described in Materials and Methods. Mitochondria were split into equal aliquots and diluted into osmotically supportive buffer (mitochondria) or nonsupportive buffer (mitoplasts), which selectively ruptures the outer membrane. These samples were further diluted into three equivalent aliquots and treated with buffer, proteinase K (100 μg/ml), or proteinase K-1% Triton X-100. Equivalent amounts of all samples were resolved with SDS-PAGE and subjected to Western blotting with antisera as described previously. Fis1 was used as an outer membrane protein control.