The nuclear gene MRS2 is essential for the excision of group II introns from yeast mitochondrial transcripts in vivo.

RNA splicing defects in mitochondrial intron mutants can be suppressed by a high dosage of several proteins encoded by nuclear genes. In this study we report on the isolation, nucleotide sequence, and possible functions of the nuclear MRS2 gene. When present on high copy number plasmids, the MRS2 gene acts as a suppressor of various mitochondrial intron mutations, suggesting that the MRS2 protein functions as a splicing factor. This notion is supported by the observations that disruption of the single chromosomal copy of the MRS2 gene causes (i) a pet- phenotype and (ii) a block in mitochondrial RNA splicing of all four mitochondrial group II introns, some of which are efficiently self-splicing in vitro. In contrast, the five group I introns monitored here are excised from pre-mRNA in a MRS2-disrupted background although at reduced rates. So far the MRS2 gene product is unique in that it is essential for splicing of all four group II introns, but relatively unimportant for splicing of group I introns. In strains devoid of any mitochondrial introns the MRS2 gene disruption still causes a pet- phenotype and cytochrome deficiency, although the standard pattern of mitochondrial translation products is produced. Therefore, apart from RNA splicing, the absence of the MRS2 protein may disturb the assembly of mitochondrial membrane complexes.     

Characterization of a novel human putative mitochondrial transporter homologous to the yeast mitochondrial RNA splicing proteins 3 and 4

We report here a novel human gene, hMRS3/4, encoding a putative mitochondrial transporter structurally and functionally homologous to the yeast mitochondrial RNA splicing proteins 3 and 4. These proteins belong to the family of mitochondrial carrier proteins (MCF) and are likely to function as solute carriers. hMRS3/4 spans approximately 10 kb of genomic DNA on chromosome 10q24 and consists of four exons that encode a 364-aa protein with six transmembrane domains. A putative splice variant, encoding a 177-aa protein with three transmembrane domains, was also identified. hMRS3/4 has a well-conserved signature sequence of MCF and is targeted into the mitochondria. When expressed in yeast, hMRS3/4 efficiently restores the mitochondrial functions in mrs3(o)mrs4(o) knock-out mutants. Ubiquitous expression in human tissues and a well-conserved structure and function suggest an important role for hMRS3/4 in human cells.     

A specific role of the yeast mitochondrial carriers MRS3/4p in mitochondrial iron acquisition under iron-limiting conditions

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the "iron regulon." We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.     

Differential Requirements for Alternative Splicing and Nuclear Export Functions of Equine Infectious Anemia Virus Rev Protein

The Rev protein of equine infectious anemia virus (ERev) exports unspliced and partially spliced viral RNAs from the nucleus. Like several cellular proteins, ERev regulates its own mRNA by mediating an alternative splicing event. To determine the requirements for these functions, we have identified ERev mutants that affect RNA export or both export and alternative splicing. Mutants were further characterized for subcellular localization, nuclear-cytoplasmic shuttling, and multimerization. None of the nuclear export signal (NES) mutants are defective for alternative splicing. Furthermore, the NES of ERev is similar in composition but distinct in spacing from other leucine-rich NESs. Basic residues at the C terminus of ERev are involved in nuclear localization, and disruption of the C-terminal residues affects both functions of ERev. ERev forms multimers, and no mutation disrupts this activity. In two mutants with substitutions of charged residues in the middle of ERev, RNA export is affected. One of these mutants is also defective for ERev-mediated alternative splicing but is identical to wild-type ERev in its localization, shuttling, and multimerization. Together, these results demonstrate that the two functions of ERev both require nuclear import and at least one other common activity, but RNA export can be separated from alternative splicing based on its requirement for a functional NES.     

A yeast nuclear gene, MRS1, involved in mitochondrial RNA splicing: nucleotide sequence and mutational analysis of two overlapping open reading frames on opposite strands.

We have cloned a 1.6-kb fragment of yeast nuclear DNA, which complements pet- mutant MK3 (mrs1). This mutant was shown to be defective in mitochondrial RNA splicing: the excision of intron 3 from the mitochondrial COB pre-RNA is blocked. The DNA sequence of the nuclear DNA fragment revealed two open reading frames (ORF1 with 1092 bp; ORF2 with 735 bp) on opposite strands, which overlap by 656 bp. As shown by in vitro mutagenesis, ORF1, but not ORF2, is responsible for complementation of the splice defect. Hence, ORF1 represents the nuclear MRS1 gene. Disruption of the gene (both ORFs) in the chromosomal DNA of the respiratory competent yeast strain DBY747 (long form COB gene) leads to a stable pet- phenotype and to the accumulation of the same mitochondrial RNA precursors as in strain MK3. The amino acid sequence of the putative ORF1 product does not exhibit any homology with other known proteins, except for a small region of homology with the gene product of another nuclear yeast gene involved in mitochondrial RNA splicing, CBP2. The function of the MRS1 (ORF1) gene in mitochondrial RNA splicing and the significance of the overlapping ORFs in this gene are discussed.     

Deletion of the mitochondrial carrier genes MRS3 and MRS4 suppresses mitochondrial iron accumulation in a yeast frataxin-deficient strain

The mitochondrial solute carriers Mrs3p and Mrs4p were originally isolated as multicopy suppressors of intron splicing defects. We show here that MRS4 is co-regulated with the iron regulon genes, and up-regulated in a strain deficient for Yfh1p, the yeast homologue of human frataxin. Using in vivo 55Fe cell radiolabeling we show that in glucose-grown cells mitochondrial iron accumulation is 5-15 times higher in deltaYFH1 than in wild-type strain. However, although in a deltaYFH1deltaMRS3deltaMRS4 strain, the intracellular 55Fe content is extremely high, the mitochondrial iron concentration is decreased to almost wild-type levels. Moreover, deltaYFH1deltaMRS3deltaMRS4 cells grown in high iron media do not lose their mitochondrial genome. Conversely, a deltaYFH1 strain overexpressing MRS4 has an increased mitochondrial iron content and no mitochondrial genome. Therefore, MRS4 is required for mitochondrial iron accumulation in deltaYFH1 cells. Expression of the iron regulon and intracellular 55Fe content are higher in a deltaMRS3deltaMRS4 strain than in the wild type. Nevertheless, the mitochondrial 55Fe content, a balance between iron uptake and exit, is decreased by a factor of two. Moreover, 55Fe incorporation into heme by ferrochelatase is increased in an MRS4-overexpressing strain. The function of MRS4 in iron import into mitochondria is discussed.     

Cardiolipin,a critical determinant of mitochondrial carrier protein assembly and function

The ability of phospholipids to act as determinants of membrane protein structure and function is probably best exemplified by cardiolipin (CL), the signature phospholipid of mitochondria. Early efforts to reconstitute individual respiratory complexes and members of the mitochondrial carrier family, most notably the ADP/ATP carrier (AAC), often demonstrated the importance of CL. Over the past decade, the significance of CL in the organization of components of the electron transport chain into higher order assemblies, termed respiratory supercomplexes, has been established. Another protein required for oxidative phosphorylation, AAC, has received comparatively little attention likely stemming from the fact that AACs were thought to function in isolation as either homodimers or monomers. Recently however, AACs have been demonstrated to interact with the respiratory supercomplex, other members of the mitochondrial carrier family, and the TIM23 translocon. Interestingly, many if not all of these interactions depend on CL. As the paradigm for the mitochondrial carrier family, these discoveries with AAC suggest that other members of this large group of important proteins may be more gregarious than anticipated. Moreover, it is proposed that AAC and perhaps additional members of the mitochondrial carrier family might represent downstream targets of pathological states involving alterations in CL.     

Mutational analysis of functional domains in Mrs2p, the mitochondrial Mg2+ channel protein of Saccharomyces cerevisiae

The nuclear gene MRS2 in Saccharomyces cerevisiae encodes an integral protein (Mrs2p) of the inner mitochondrial membrane. It forms an ion channel mediating influx of Mg2+ into mitochondria. Orthologues of Mrs2p have been shown to exist in other lower eukaryotes, in vertebrates and in plants. Characteristic features of the Mrs2 protein family and the distantly related CorA proteins of bacteria are the presence of two adjacent transmembrane domains near the C terminus of Mrs2p one of which ends with a F/Y-G-M-N motif. Two coiled-coil domains and several conserved primary sequence blocks in the central part of Mrs2p are identified here as additional characteristics of the Mrs2p family. Gain-of-function mutations obtained upon random mutagenesis map to these conserved sequence blocks. They lead to moderate increases in mitochondrial Mg2+ concentrations and concomitant positive effects on splicing of mutant group II intron RNA. Site-directed mutations in several conserved sequences reduce Mrs2p-mediated Mg2+ uptake. Mutants with strong effects on mitochondrial Mg2+ concentrations also have decreased group II intron splicing. Deletion of a nonconserved basic region, previously invoked for interaction with mitochondrial introns, lowers intramitochondrial Mg2+ levels as well as group II intron splicing. Data presented support the notion that effects of mutations in Mrs2p on group II intron splicing are a consequence of changes in steady-state mitochondrial Mg2+ concentrations.     

Yeast ADP/ATP carrier (AAC) proteins exhibit similar enzymatic properties but their deletion produces different phenotypes.

Saccharomyces cerevisiae strains expressing a single type of ADP/ATP carrier (AAC) protein were prepared from a mutant in which all AAC genes were disrupted, by transformation with plasmids containing a chosen AAC gene. As demonstrated by measurements of [14C]ADP specific binding and transport, all three translocator proteins, AAC1, AAC2 and AAC3 when present in the mitochondrial membrane, exhibited similar translocation properties. The disruption of some AAC genes, however, resulted in phenotypes indicating that the function of these proteins in whole cells can be quite different. Specifically, we found that the disruption of AAC1 gene, but not AAC2 and AAC3, resulted in a change in colony phenotype.     

Presence of a member of the mitochondrial carrier family in hydrogenosomes: conservation of membrane-targeting pathways between hydrogenosomes and mitochondria

A number of microaerophilic eukaryotes lack mitochondria but possess another organelle involved in energy metabolism, the hydrogenosome. Limited phylogenetic analyses of nuclear genes support a common origin for these two organelles. We have identified a protein of the mitochondrial carrier family in the hydrogenosome of Trichomonas vaginalis and have shown that this protein, Hmp31, is phylogenetically related to the mitochondrial ADP-ATP carrier (AAC). We demonstrate that the hydrogenosomal AAC can be targeted to the inner membrane of mitochondria isolated from Saccharomyces cerevisiae through the Tim9-Tim10 import pathway used for the assembly of mitochondrial carrier proteins. Conversely, yeast mitochondrial AAC can be targeted into the membranes of hydrogenosomes. The hydrogenosomal AAC contains a cleavable, N-terminal presequence; however, this sequence is not necessary for targeting the protein to the organelle. These data indicate that the membrane-targeting signal(s) for hydrogenosomal AAC is internal, similar to that found for mitochondrial carrier proteins. Our findings indicate that the membrane carriers and membrane protein-targeting machinery of hydrogenosomes and mitochondria have a common evolutionary origin. Together, they provide strong evidence that a single endosymbiont evolved into a progenitor organelle in early eukaryotic cells that ultimately give rise to these two distinct organelles and support the hydrogen hypothesis for the origin of the eukaryotic cell.     

Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier.

We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein. The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively. The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids. The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively. Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure. Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.     

Structure-function studies of adenine nucleotide transport in mitochondria. II. Biochemical analysis of distinct AAC1 and AAC2 proteins in yeast

AAC1 and AAC2 genes in yeast each encode functional ADP/ATP carrier (AAC) proteins of the mitochondrial inner membrane. In the present study, mitochondria harboring distinct AAC proteins and the pet9 Arg96 to HIS mutant (Lawson, J., Gawaz, M., Klingenberg, M., and Douglas, M. G. (1990) J. Biol. Chem. 265, 14195-14201) protein have been characterized. In addition, properties of the different AAC proteins have been defined following reconstitution into proteoliposomes. Deletion of AAC2 but not AAC1 causes a major reduction in the mitochondrial cytochrome content and respiration, and this level remains low even when the level of AAC1 protein is increased to 20% that of the AAC2 gene product. In reconstitution studies, the rate of nucleotide transport by isolated AAC1 protein is approximately 40% that of the AAC2 protein. Thus, the lack of mitochondrial-dependent growth supported by the AAC1 gene product alone may be due to the combination of low abundance and reduced activity. Surprisingly, analysis of the Arg96 to His mutant protein revealed binding and transport activities similar to the functional AAC1 and AAC2 gene products. These observations are discussed in relation to a molecular analysis of this highly conserved small transporter and its function in conjunction with other proteins in the mitochondrial membrane.     

Arginine/serine repeats are sufficient to constitute a splicing activation domain

SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine–serine (RS) rich domain. The RS domains mediate protein–protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.     

Biogenesis of the dicarboxylate carrier (DIC): translocation across the mitochondrial outer membrane and subsequent release from the TOM channel are membrane potential-independent

The mitochondrial inner membrane of Saccharomyces cerevisiae contains a group of homologous carrier proteins that mediate the exchange of several metabolites. The members of this protein family are synthesized in the cytosol and reach their final topology after translocation across the mitochondrial outer membrane. Using the ADP/ATP carrier (AAC) as a model protein, previous studies have established four distinct steps of the import pathway (stages I-IV). In the absence of the mitochondrial membrane potential (deltapsi), the AAC accumulates at the inner surface of the outer membrane (stage IIIa) and remains bound to the outer membrane import channel. Only in the presence of the membrane potential, can a complex of small Tim proteins mediate transfer of the AAC to the inner membrane. In this study, we characterized the import pathway of the dicarboxylate carrier (DIC). Different from the AAC, the DIC showed complete deltapsi-independent translocation across the outer membrane, release from the import pore, and mainly accumulated in a soluble state in the intermembrane space, thus defining a new translocation intermediate (stage III*). The DIC should be a suitable model protein for the characterization of deltapsi-independent functions of the intermembrane space Tim proteins.     

Molecular aspects of the adenine nucleotide carrier from mitochondria

The ADP/ATP carrier (AAC) of mitochondria is a functionally central and characteristic component of the eukaryotic cell. By linking the thermodynamically divergent metabolites in the intra- and extramitochondrial compartments, it had to evolve with the emergence of the eukaryotic cell. Because of a number of unique properties, the AAC provided advanced insight into the molecular basis of solute transport through biomembrane carriers. With highly specific and unusually large substrates, ADP and ATP, and with high-affinity inhibitors binding selectively either from the inside or the outside, the first molecular demonstration of the single-binding-center gated pore mechanism was made. This framework can only partially be interpreted with the available yet rapidly increasing structural information on the AAC. The primary structure, first established for the AAC from beef heart mitochondria, showed a relatively wide distribution of hydrophilic residues which permits assignment of only two hydrophobic transmembrane stretches. However, a striking tripartition of the primary structure into about three 100-residue-long domains allows a more significant assignment of transmembrane elements. With alignment of these three domains for maximum conservation of structurally critical residues, each domain can be assigned to have two transmembrane alpha elements between 18 and 22 residues long. The interdomain homology between these alpha regions is low. The central regions flanked by these helices contain most of the polar residues and are significantly interdomain conserved. With lysine probes the central regions are assigned to the matrix side (m-side) and the two connecting regions as well as C and N termini to the cytosolic side (c-side). Out of the central regions a loop is assumed to protrude through the membrane, probably for lining the translocation channel. This localization of a major protein mass within the membrane agrees with hydrodynamic evidence, the carrier being an oblate ellipsoid with only about 50 A along the short axis. In accordance, the loops of domains 2 and 3 are affinity labeled by azido-ADP or azido-atractylate. Primary structures of AAC from other sources (fungi, plants) also exhibit the tripartition. The interdomain conserved residues are also interspecies conserved, thus showing that they are essential. These repeat domains have probably evolved from a common gene coding for about 100 residues. Isoforms of the AAC exist, as shown by primary structure analysis of human cDNA libraries from different organs. Three different isoforms are identified in human organs.(ABSTRACT TRUNCATED AT 400 WORDS)