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.

Figure 1.

MLR proteins of seven mitochondrial complexes. The association between mitochondrial-bound polysomes and 107 mRNAs coding for all the subunits of mitochondrial complexes: respiratory complexes 2, 3, 4, and 5; import complexes TOM and TIM; and TCA cycle complexes, and for assembly factors (starred) was quantified by RT-PCR analyses with several correction factors (see Materials and Methods and Garcia et al., 2006). Only the genes that are translated in the vicinity of mitochondria (MLR proteins) are represented (all data are available in Supplementary Material). Most of these MLR genes have prokaryotic homologues (gray shaded box), whereas most of the other genes have no known prokaryotic homologue (Supplementary Material). The right column gives a schematic composition of the assembled complexes according to gene origin: mitochondrially encoded (white), nuclearly encoded either of prokaryotic (dark gray), or eukaryotic (light gray) origin.

Figure 2.

Live-cell imaging of nuclear-encoded mRNAs for mitochondrial proteins. Five fluorescent oligonucleotides specific for mitochondrial rRNA (lane B) and for specific mRNAs (ATP16-C1, ATP3-C2, ATP2-C3, ATP4-C4, and TIM50-C5) were used to probe wild-type yeast cells for hybridization. The merged figures (lane D) indicate the relative localizations of mt rRNA (red) and the various mRNAs (green). 3D quantitative analysis was used to compare ATP16 mRNA, which is isotropically distributed, with the specifically localized ATP3 mRNA (Supplementary Figure 2S). A 3D reconstitution of these relative localizations is represented on the bottom line for ATP2 mRNA (green), mt rRNA (violet), and the negative control YRA1 mRNA (red). These 3D analyses allow a quantitative assessment of the distance between a particular mRNA molecule and the edge of the mitochondrion (see Supplementary Data).

Figure 3.

Class I intermembrane space proteins are MLR proteins. Protein-translocation pathways into the intermembrane space (IMS) of mitochondria have been classified into three classes (Herrmann and Hell, 2005). Class I proteins have typical mitochondrial presequences followed by hydrophobic sorting domains and are directed to the TIM23 complex. Class II and III proteins are imported independently of the inner-membrane TIM23 complex. Polysome localization of mRNA coding for the IMS proteins described was examined by RT-PCR analysis. Class I proteins were clearly produced from mRNA translated in the vicinity of mitochondria (MLR proteins-RT-PCR value above 6%, indicated in dark gray in the right column), whereas none of the mRNAs for the class II or III proteins were significantly linked to mitochondria (non-MLR proteins, white; RT-PCR value <6%).

Figure 4.

Proposed model of inner-membrane mitochondrial complex biogenesis. Three types of proteins compose the inner-membrane complexes: (1) Mitochondrial-encoded proteins translated on membrane-bound mRNA, depending on specific activator proteins. (2) MLR proteins, identified in this work, are nuclear-encoded and translated in the vicinity of the outer mitochondrial membrane. (3) Nuclear-encoded proteins translated on free cytoplasmic polysomes and that are posttranslationally imported. The model assumes that pathways 1 and 2 are spatially connected, thus facilitating the formation of the core protein complex with which accessory proteins (pathway 3) will combine.