Abstract
The amino-terminal domain (ATD) of Saccharomyces cerevisiae mitochondrial RNA polymerase has been shown to provide a functional link between transcription and post-transcriptional events during mitochondrial gene expression. This connection is mediated in large part by its interactions with the matrix protein Nam1p and, based on genetic phenotypes, the mitochondrial membrane protein Sls1p. These observations led us to propose previously that mtRNA polymerase, Nam1p, and Sls1p work together to coordinate transcription and translation of mtDNA-encoded gene products. Here we demonstrate by specific labeling of mitochondrial gene products in vivo that Nam1p and Sls1p indeed work together in a pathway that is required globally for efficient mitochondrial translation. Likewise, mutations in the ATD result in similar global reductions in mitochondrial translation efficiency and sensitivity to the mitochondrial translation inhibitor erythromycin. These data, coupled with the observation that the ATD is required to co-purify Sls1p in association with mtDNA nucleoids, suggest that efficient expression of mtDNA-encoded genes in yeast involves a complex series of interactions that localize active transcription complexes to the inner membrane in order to coordinate translation with transcription.
Highlights
The ϳ80-kb Saccharomyces cerevisiae mitochondrial genome encodes seven oxidative phosphorylation subunits destined for the inner mitochondrial membrane, one ribosomal protein, two rRNAs, and a full complement of tRNAs [1]
Because nam1⌬ and amino-terminal domain (ATD) mutant strains all demonstrate a defect in intron processing of the COX1 and COB genes [10, 15] and Sls1p has been implicated to act in the same pathway of transcription-coupled events as Nam1p and mtRNA polymerase [9], we investigated whether an sls1⌬ strain has a similar mitochondrial intron-processing defect as nam1⌬ and rpo41 ATD mutant strains
This is a complex process that involves membrane-associated ribosomes and mRNA-specific translational activators and numerous other proteins involved in processing and stability of mRNA that are directly or indirectly associated with the inner membrane [4, 7]
Summary
Media—Yeast were grown as described [19] in complete YPG (glycerol) medium, YPD (dextrose) medium, or synthetic dextrose (SD) medium with the necessary nutritional supplements as indicated. Radiolabeling of Mitochondrial Translation Products in Vivo—An overnight culture (2 ml) of each yeast strain was grown to saturation in YPG (or YPD for petite mutants) medium and used to inoculate a 15-ml SD culture supplemented with the appropriate amino acids. These cultures were incubated at 30 °C for 2 h prior to overnight incubation at 37 °C. Mitochondria were resuspended in 1 ml of ice-cold protein synthesis medium, and pyruvate kinase was added, and the sample was incubated at room temperature for 10 min prior to the addition of 400 M of the cross-linking reagent dithiobis(succinimidyl propionate) (Pierce). A sample of each nucleoid fraction (40 l) and each lysate sample (20 l) were separated on an 8% SDS-PAGE gel and transferred to a membrane for Western analysis using an anti-V5 antibody (Invitrogen)
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