Mitochondria-associated Yeast mRNAs and the Biogenesis of Molecular Complexes

Abstract

The coherence of mitochondrial biogenesis relies on spatiotemporally coordinated associations of 800 -1000 proteins mostly encoded in the nuclear genome. We report the development of new quantitative analyses to assess the role of local protein translation in the construction of molecular complexes. We used real-time PCR to determine the cellular location of 112 mRNAs involved in seven mitochondrial complexes. Five typical cases were examined by an improved FISH protocol. The proteins produced in the vicinity of mitochondria (MLR proteins) were, almost exclusively, of prokaryotic origin and are key elements of the core construction of the molecular complexes; the accessory proteins were translated on free cytoplasmic polysomes. These two classes of proteins correspond, at least as far as intermembrane space (IMS) proteins are concerned, to two different import pathways. Import of MLR proteins involves both TOM and TIM23 complexes whereas non-MLR proteins only interact with the TOM complex. Site-specific translation loci, both outside and inside mitochondria, may coordinate the construction of molecular complexes composed of both nuclearly and mitochondrially encoded subunits. INTRODUCTIONMost mitochondrial proteins of eukaryotic cells are encoded by nuclear genes and synthesized by cytoplasmic ribosomes. However, a few proteins are encoded by the mitochondrial DNA and are translated inside mitochondria by bacterialtype ribosomes. The major mitochondrial import pathway of cytoplasmically translated proteins is now well understood (Koehler, 2004;Rehling et al., 2004). Although a posttranslational mechanism for import is widely accepted, it may concern only a limited class of proteins. Indeed, it was demonstrated 30 years ago that a subclass of cytoplasmic polysomes is bound to the surface of mitochondria (Kellems et al., 1975). Subsequently, experiments in yeast and human cells have suggested a cotranslational import process for some mitochondrial proteins (Fujiki and Verner, 1993;Mukhopadhyay et al., 2004). The evolutionary history of mitochondria might elucidate this apparent discrepancy between post-and cotranslational import processes. Phylogenetic studies of the yeast mitochondrial proteome have demonstrated a composite origin. It is estimated that half or more of the genes that code for the modern mitochondrial proteome originated directly from the host nuclear genome, and the other half are of bacterial origin, a consequence of a massive transfer of genes from the endosymbiont to the host nuclear genome (Marcotte et al., 2000;Karlberg et al., 2000). This symbiotic situation required the development of protein translocation machineries (translocases) in the mitochondrial membranes (Herrmann, 2003). Possibly, the two classes of nuclear genes coding for mitochondrial products may have used the same mitochondrial import pathway. However, a recent genomewide analysis (Marc et al., 2002) suggested a strong correlation between the bacterial origin of the genes and their locus specific translation on mitochondria-l...

Localization of mRNAs coding for mitochondrial proteins in the yeast Saccharomyces cerevisiae

“…About half of all mMPs are thought to be translated in the vicinity of mitochondria and mRNAs of this class were named mitochondrially localized mRNAs (MLRs) (Marc et al 2002). This suggested that mitochondrial protein import could be cotranslational and indicated that MLR proteins are mainly of prokaryotic origin, linked to the assembly of core complexes, and are imported principally via the TOM-TIM23 pathway (Garcia et al 2007). Puf3, an RNA-binding protein (RBP) that belongs to the Pumilio-Fbf (Puf) family and interacts with more than 150 mMPs (Gerber et al 2004), was recently shown to be involved in the transport of mMPs encoding proteins involved in respiration and translational control within the organelle; and the loss of PUF3 gene expression influenced mRNA association with the mitochondria (SaintGeorges et al 2008).…”

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

“…These studies where then corroborated in vivo by work on specific mRNAs, that were found to be imported only in a cotranslational manner 6,7 . Genome-wide studies of mRNAs association with mitochondria revealed that a significant fraction of mRNAs are localized to the mitochondria vicinity [8][9][10] . Some of these mRNAs were further characterized by in vivo fluorescence methods, such as FISH or mTAG 9,11 .…”

“…Genome-wide studies of mRNAs association with mitochondria revealed that a significant fraction of mRNAs are localized to the mitochondria vicinity [8][9][10] . Some of these mRNAs were further characterized by in vivo fluorescence methods, such as FISH or mTAG 9,11 . A straight-forward interpretation of this association is that these mRNAs serve as templates for localized translation.…”

“…Localized translation near the mitochondria was studied by various methods, including electron microscopy (to visualize ribosomes) 3 , FISH 9 , green RNA (to detect specific mRNAs) 11 , and biochemical fractionation (to detect both RNA and ribosomes) 10,18 . While the former methods detect localization in vivo and may allow visualization of transport dynamics, the latter allows detection of many different mRNAs in a single experiment.…”

Single yeast cells vary in transcription activity not in delay time after a metabolic shift

“…Since the lifetime of fluorescent reporter proteins is typically not the same as the protein it is reporting, the applicability of fluorescent reporter proteins to study transcription repression is limited. Singlemolecule RNA FISH has been applied to yeast before to address a wide variety of transcription and cell-to-cell heterogeneity-related questions [27][28][29][30][31][32][33][34] .…”