Translational Regulation of Nuclear Gene COX4 Expression by Mitochondrial Content of Phosphatidylglycerol and Cardiolipin in Saccharomyces cerevisiae

Cardiolipin (CL) and its precursor phosphatidylglycerol (PG) are anionic phospholipids whose synthesis and distribution are limited primarily to the mitochondrial inner membrane of eukaryotic cells (25). As the major anionic phospholipid in the mitochondrial inner membrane, CL is specifically associated with a number of mitochondrial proteins, many of which also require CL for their optimal functions (4, 12, 16, 26, 32, 55). Together with PG, CL has been postulated or demonstrated to participate in many critical mitochondrial processes, such as maintaining membrane permeability and integrity (25, 33), solute transport (4, 31, 47), oxidative phosphorylation (35, 72), protein and phospholipid import (2, 13), and mitochondrion-mediated apoptosis (46). Due to its highly unsaturated fatty acyl chains, CL is considered an “antioxidant” and the primary lipid peroxidation target in mitochondria. Reduction in CL levels by oxidative damage or other means is correlated with negative aspects of several physiological states, such as Barth's syndrome (67), ischemia/reperfusion (41), and aging (50, 60). Saccharomyces cerevisiae mutants disrupted in the PGS1 (encodes phosphatidylglycerophosphate synthase) (7) or CRD1 (encodes CL synthase) (8, 30, 64) gene have been created, and both manifest growth defects on nonfermentable carbon sources, indicating a central role for these lipids in oxidative phosphorylation. Yeast mutants (crd1Δ) deficient only in CL display a 7- to 10-fold increase in PG that may partially complement some of the CL functions, thereby masking the full effect of lack of these anionic phospholipids. Despite complementation by PG, CL has been shown to be essential under stress conditions (73) or for efficient coupling between complexes III and IV of the respiratory chain (52, 72). Yeast mutants (pgs1Δ) deficient in both PG and CL display more severe defects in mitochondrial function, such as slow growth on a fermentable carbon source and total inability to respire on nonfermentable carbon sources. One of the reasons for the lack of growth of pgs1Δ cells on nonfermentable carbon sources appears to be due to an initial reduction in the level of respiratory components (48) and eventual loss of mitochondrial DNA (mtDNA) (74). In yeast, cytochrome c oxidase (complex IV) is a multisubunit complex consisting of 11 subunits (19). Subunits I, II, and III (Cox1p to Cox3p), comprising the catalytic core of the complex, are encoded by the mitochondrial genome, with the remainder, including subunit IV (Cox4p), encoded by the nuclear genome. In pgs1Δ cells, four of mitochondrion-encoded proteins (Cox1p to Cox3p and Cobp, a subunit of cytochrome bc1 (complex III)) and one nucleus-encoded protein (Cox4p) were reported missing (48). Although previous results were consistent with a block in translation of COX4 mRNA, rapid degradation of the unassembled components of complex IV (11) in the absence of the essential structural component, CL (57), was not completely excluded. If lack of expression of nucleus-encoded Cox4p is due to a block in translation in pgs1Δ cells, how can the cytoplasmic translational machinery “sense” changes in mitochondrial phospholipid composition and repress translation of certain proteins that are targeted to mitochondria, where they are assembled into oxidative complexes together with the mitochondrion-encoded subunits? Cross talk between mitochondria and the nucleus is well documented (5, 15, 34, 51, 54). Mitochondrial stress such as mtDNA mutations and inhibition of respiration or mitochondrial gene expression can serve as signals transmitted to the nucleus to induce the expression of specific nuclear genes that in turn help mitochondria cope with stress (3, 14, 53). Most of this type of interorganelle signaling studied to date has involved regulation at the transcriptional level, but the mRNAs of some nucleus-encoded mitochondrial proteins are translated while bound to the mitochondrial membrane, possibly making their translation sensitive to mitochondrial membrane lipid composition. However, COX4 was not identified within the subset of nuclear genes which displayed a high value for mitochondrial localization of mRNA (MLR) in a genome-wide DNA microarray analysis (45), even though import of Cox4p into mitochondria is greatly enhanced cotranslationally compared to posttranslationally in a homologous yeast in vitro system (1, 17, 18). The MLR value for COX4 is 26.7 (on a scale of 100), similar to POR1 (encodes mitochondrial porin), with a MLR value of 19, suggesting that the majority of COX4 mRNA may be translated on free ribosomes in the cytoplasm. In this study, we focused on the mechanism of repression of nucleus-encoded Cox4p expression in response to changes in mitochondrial anionic phospholipid content. Cox4p mRNA contains an intron in the 5′ untranslated region (UTR) (56) and encodes a cytoplasmically translated protein product with an amino-terminal mitochondrial targeting sequence. Our results establish suppression of Cox4p translational in response to lack of mitochondrial PG and CL (pgs1Δ cells) and support a novel cross talk mechanism, which regulates cytoplasmic translation of COX4 mRNA through binding of a nucleus-encoded trans-acting repressor protein(s) to a sequence in the 5′ UTR of the mature mRNA.