Mdm38 protein depletion causes loss of mitochondrial K+/H+ exchange activity, osmotic swelling and mitophagy.

Loss of the MDM38 gene product in yeast mitochondria results in a variety of phenotypic effects including reduced content of respiratory chain complexes, altered mitochondrial morphology and loss of mitochondrial K(+)/H(+) exchange activity resulting in osmotic swelling. By use of doxycycline-regulated shut-off of MDM38 gene expression, we show here that loss of K(+)/H(+) exchange activity and mitochondrial swelling are early events, associated with a reduction in membrane potential and fragmentation of the mitochondrial reticulum. Changes in the pattern of mitochondrially encoded proteins are likely to be secondary to the loss of K(+)/H(+) exchange activity. The use of a novel fluorescent biosensor directed to the mitochondrial matrix revealed that the loss of K(+)/H(+) exchange activity was immediately followed by morphological changes of mitochondria and vacuoles, the close association of these organelles and finally uptake of mitochondrial material by vacuoles. Nigericin, a K(+)/H(+) ionophore, fully prevented these effects of Mdm38p depletion. We conclude that osmotic swelling of mitochondria triggers selective mitochondrial autophagy or mitophagy.

Mitochondrial Mg(2+) homeostasis is critical for group II intron splicing in vivo.

The product of the nuclear MRS2 gene, Mrs2p, is the only candidate splicing factor essential for all group II introns in mitochondria of the yeast Saccharomyces cerevisiae. It has been shown to be an integral protein of the inner mitochondrial membrane, structurally and functionally related to the bacterial CorA Mg(2+) transporter. Here we show that mutant alleles of the MRS2 gene as well as overexpression of this gene both increase intramitochondrial Mg(2+) concentrations and compensate for splicing defects of group II introns in mit(-) mutants M1301 and B-loop. Yet, covariation of Mg(2+) concentrations and splicing is similarly seen when some other genes affecting mitochondrial Mg(2+) concentrations are overexpressed in an mrs2Delta mutant, indicating that not the Mrs2 protein per se but certain Mg(2+) concentrations are essential for group II intron splicing. This critical role of Mg(2+) concentrations for splicing is further documented by our observation that pre-mRNAs, accumulated in mitochondria isolated from mutants, efficiently undergo splicing in organello when these mitochondria are incubated in the presence of 10 mM external Mg(2+) (mit(-) M1301) and an ionophore (mrs2Delta). This finding of an exceptional sensitivity of group II intron splicing toward Mg(2+) concentrations in vivo is unprecedented and raises the question of the role of Mg(2+) in other RNA-catalyzed reactions in vivo. It explains finally why protein factors modulating Mg(2+) homeostasis had been identified in genetic screens for bona fide RNA splicing factors.

The human mitochondrial Mrs2 protein functionally substitutes for its yeast homologue, a candidate magnesium transporter.

We report here on the human MRS2 gene that encodes a protein, hsaMrs2p, the first molecularly characterized candidate for a magnesium transporter in metazoa. The protein, like the yeast mitochondrial Mrs2 and Lpe10 proteins, contains two predicted transmembrane domains in its carboxyl-terminus, the first of which terminates with the conserved motif F/Y-G-M-N. These are typical features of the CorA family of magnesium transporters. Expression of hsaMrs2p in mrs2-1 knock-out mutant yeast partly restores mitochondrial magnesium concentrations that are significantly reduced in this mutant. It also alleviates other defects of this mutant, which may be secondary to the reduction in magnesium concentrations. These findings suggest that hsaMrs2p and yMrs2p are functional homologues. Like its yeast homologues, hsaMrs2p has been localized in mitochondria. The hsaMRS2 gene is located on chromosome 6 (6p22.1-p22.3) and is composed of 11 exons. A low level of the transcript is detected in various mouse tissues.

Ypt protein prenylation depends on the interplay among levels of Rab escort protein and geranylgeranyl diphosphate in yeast cells.

Farnesyl diphosphate (FPP), an intermediate of the sterol biosynthetic pathway, is used by farnesyl transferase to farnesylate, among others, the Ras proteins, and by geranylgeranyl diphosphate synthase to produce geranylgeranyl diphosphate (GGPP). GGPP is then transferred by geranylgeranyl transferase II (GGTase II) to Rab/Ypt members of the Ras superfamily known to be required at all stages of vesicle transport in both mammals and yeast. Formation of a complex between a Rab/Ypt protein and an accessory protein named the Rab escort protein (REP) is a prerequisite for GGTase II substrate recognition. Little is known about the factors that regulate GGTase II activity in living cells but, based on available data, it seems possible that vesicle transport in higher eukaryotes is regulated by the levels of prenylated Rab/Ypt proteins in the cells. Here we show that the levels of REP play an important role in regulating GGTase II activity in yeast cells if sufficient substrates are present. Moreover, overexpression of REP causes, directly or indirectly, an increased level of Ypt substrates available for prenylation, which in turn leads to the depletion of the GGPP pool in the cell. Overall our data suggest that the levels of REP and the availability of GGPP play a role in regulating Ypt protein prenylation.

The mitochondrial inner membrane protein Lpe10p, a homologue of Mrs2p, is essential for magnesium homeostasis and group II intron splicing in yeast.

The yeast ORF YPL060w/LPE10 encodes a homologue of the mitochondrial protein Mrs2p. These two proteins are 32% identical, and have two transmembrane domains in their C-terminal regions and a putative magnesium transporter signature, Y/F-G-M-N, at the end of one of these domains. Data presented here indicate that Lpe10p is inserted into the inner mitochondrial membrane with both termini oriented towards the matrix space. Disruption of the LPE10 gene results in a growth defect on non-fermentable substrates (petite phenotype) and a marked defect in group II intron splicing. The fact that in intron-less strains lpe10 disruptants also exhibit a petite phenotype indicates that functions other than RNA splicing are affected by the absence of Lpe10p. In the mitochondria, concentrations of magnesium, but not of several other divalent metal ions, are increased when Lpe10p is overexpressed and reduced when it is absent. Magnesium concentrations are raised to normal levels and growth on non-fermentable substrates is partially restored by the expression of CorA, the bacterial magnesium transporter, in the lpe10 disruptant. These features are similar to those previously reported for Mrs2p, suggesting that Lpe10p and Mrs2p are functional homologues. However, they cannot easily substitute for each other. Their roles in magnesium homeostasis and, possibly as a secondary effect, in RNA splicing are discussed.

The yeast plasma membrane protein Alr1 controls Mg2+ homeostasis and is subject to Mg2+-dependent control of its synthesis and degradation.

The Saccharomyces cerevisiae ALR1 (YOL130w) gene product Alr1p is the first known candidate for a Mg(2+) transport system in eukaryotic cells and is distantly related to the bacterial CorA Mg(2+) transporter family. Here we provide the first experimental evidence for the location of Alr1p in the yeast plasma membrane and for the tight control of its expression and turnover by Mg(2+). Using well characterized npi1 and end3 mutants deficient in the endocytic pathway, we demonstrate that Alr1 protein turnover is dependent on ubiquitination and endocytosis. Furthermore, cells lacking the vacuolar protease Pep4p accumulated Alr1p in the vacuole. Mutants lacking Alr1p (Deltaalr1) showed a 60% reduction of total intracellular Mg(2+) compared with the wild type and failed to grow in standard media. When starved of Mg(2+), mutant and wild-type cells had similar low levels of intracellular Mg(2+); but upon addition of Mg(2+), wild-type cells replenished the intracellular Mg(2+) pool within a few hours, whereas Deltaalr1 mutant cells did not. Expression of the bacterial Mg(2+) transporter CorA in the yeast Deltaalr1 mutant partially restored growth in standard media. The results are discussed in terms of Alr1p being a plasma membrane transporter with high selectivity for Mg(2+).

Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance.

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.

Transposition and exon shuffling by group II intron RNA molecules in pieces.

In the realms of RNA, transposable elements created by self-inserting introns recombine novel combinations of exon sequences in the background of replicating molecules. Although intermolecular RNA recombination is a wide-spread phenomenon reported for a variety of RNA-containing viruses, direct evidence to support the theory that modern splicing systems, together with the exon-intron structure, have evolved from the ability of RNA to recombine, is lacking. Here, we used an in vitro deletion-complementation assay to demonstrate trans-activation of forward and reverse self-splicing of a fragmented derivative of the group II intron bI1 from yeast mitochondria. We provide direct evidence for the functional interchangeability of analogous but non-identical domain 1 RNA molecules of group II introns that result in trans-activation of intron transposition and RNA-based exon shuffling. The data extend theories on intron evolution and raise the intriguing possibility that naturally fragmented group III and spliceosomal introns themselves can create transposons, permitting rapid evolution of protein-coding sequences by splicing reactions.

The bacterial magnesium transporter CorA can functionally substitute for its putative homologue Mrs2p in the yeast inner mitochondrial membrane.

The yeast nuclear gene MRS2 encodes a protein of 54 kDa, the presence of which has been shown to be essential for the splicing of group II intron RNA in mitochondria and, independently, for the maintenance of a functional respiratory system. Here we show that the MRS2 gene product (Mrs2p) is an integral protein of the inner mitochondrial membrane. It appears to be inserted into this membrane by virtue of two neighboring membrane spanning domains in its carboxyl-terminal half. A large amino-terminal and a shorter carboxyl-terminal part are likely to be exposed to the matrix space. Structural features and a short sequence motif indicate that Mrs2p may be related to the bacterial CorA Mg2+ transporter. In fact, overexpression of the CorA gene in yeast partially suppresses the pet- phenotype of an mrs2 disrupted yeast strain. Disruption of the MRS2 gene leads to a significant decrease in total magnesium content of mitochondria which is compensated for by the overexpression of the CorA gene. Mutants lacking or overproducing Mrs2p exhibit phenotypes consistent with the involvement of Mrs2p in mitochondrial Mg2+ homeostasis.

Import of mitochondrial carriers mediated by essential proteins of the intermembrane space.

In order to reach the inner membrane of the mitochondrion, multispanning carrier proteins must cross the aqueous intermembrane space. Two essential proteins of that space, Tim10p and Tim12p, were shown to mediate import of multispanning carriers into the inner membrane. Both proteins formed a complex with the inner membrane protein Tim22p. Tim10p readily dissociated from the complex and was required to transport carrier precursors across the outer membrane; Tim12p was firmly bound to Tim22p and mediated the insertion of carriers into the inner membrane. Neither protein was required for protein import into the other mitochondrial compartments. Both proteins may function as intermembrane space chaperones for the highly insoluble carrier proteins.

The yeast Rab escort protein binds intracellular membranes in vivo and in vitro.

In both mammals and yeast, intracellular vesicular transport depends on the correct shuttling between membrane and cytosol of the Rab/Ypt small G proteins. Membrane association of these proteins requires prenylation by the Rab geranylgeranyl transferase that recognizes a complex formed by the Rab/Ypt protein and the Rab escort protein (REP). After prenylation the Rab/Ypt protein is delivered to the target membranes by REP. Little is known about the early steps of the Rab-REP complex formation and where this association occurs in the cell. Although prenylation is believed to take place in the cytosol, we show that the yeast Rab escort protein Mrs6 is present in both soluble and particulate fractions of cell extracts. Mrs6p is associated with the heavy microsomal fraction that contains endoplasmic reticulum-Golgi membranes but is absent in the plasma membrane, vacuoles, mitochondria, and microsomal subfraction associated with mitochondria. The solubilization pattern of the particulate pool of Mrs6p implies that this protein is peripherally but tightly associated with membranes via hydrophobic interactions and metal ions. We also report that the C terminus of Mrs6p is important for maintaining the solubility of the protein because its deletion or replacement with the C terminus of RabGDI results in a protein that localizes only to membranes.

A soluble 12-kDa protein of the mitochondrial intermembrane space, Mrs11p, is essential for mitochondrial biogenesis and viability of yeast cells.

We have isolated an essential yeast gene termed MRS11, which codes for a soluble protein of the mitochondrial intermembrane space. Interestingly, this new gene shares many similarities with the previously characterized MRS5 gene: when expressed from a multicopy plasmid, MRS11 like MRS5 restores respiration competence to yeast strains defective in the splicing of mitochondrial group II introns. Both genes are essential for viability of yeast cells, as the disruption of either of them is lethal. The proteins encoded by MRS5 and MRS11, which display 35%, sequence identity are both located in the mitochondrial intermembrane space. Depletion of Mrs11p results in a phenotype similar to that observed in Mrs5p-depleted cells: accumulation of the precursor form of mitochondrial hsp60, inability to form spectrophotometrically detectable amounts of cytochromes and changes in the mitochondrial morphology. Although similar in sequence and function, Mrs5p and Mrs11p are not functionally equivalent and neither can substitute for the other, even when overexpressed. Taken together, our data suggest a cooperative mode of action of Mrs11p and Mrs5p in mitochondrial protein import or other related essential mitochondrial processes.

DNA polymerization catalysed by a group II intron RNA in vitro.

The excised group II intron bI1 from Saccharomyces cerevisiae can act as a ribozyme catalysing various chemical reactions with different substrate RNAs in vitro . Recently, we have described an editing-like RNA polymerization reaction catalysed by the bI1 intron lariat that proceeds in the 3'-->5'direction. Here we show that the bI1 lariat RNA can also catalyse successive deoxyribonucleotide polymerization reactions on exogenous substrate molecules. The basic mechanism of the reaction involved interacting cycles between an alternative version of partial reverse splicing (lariat charging) and canonical forward splicing (lariat discharging by exon ligation). With an overall chain growth in the 3'-->5' direction, the 5' exon RNAs (IBS1dN) were elongated by successive insertion of deoxyribonucleotides derived from single deoxyribonucleotide substitutions (dA, dG, dC or dT). All four deoxyribonucleotides were used as substrates, although with different efficiencies. Our findings extend the catalytic repertoire of group II intron RNAs not only by a novel DNA polymerization activity, but also by a DNA-DNA ligation capacity, supporting the idea that ribozymes might have been part of the first primordial polymerization machinery for both RNA and DNA.

The yeast ORF YDL202w codes for the mitochondrial ribosomal protein YmL11.

The Saccharomyces cerevisiae open reading frame YDL202w has been characterised in the course of the EUROFAN yeast genome analysis program. Disruption of YDL202w causes a respiratory deficient phenotype accompanied by a loss of mitochondrial DNA. This phenotype is usually found in mutants defective in mitochondrial replication or gene expression. YDL202w has the potential to encode a soluble protein of 249 amino acids. It shows significant similarities to the ribosomal protein L10 from various bacteria and to a previously determined amino-terminal peptide sequence of the yeast mitochondrial ribosomal protein L11. The predicted amino-acid sequence of YDL202w starts with a stretch which has neither any correspondence in the bacterial sequences nor in the protein isolated from mitochondrial ribosomes. Furthermore, this stretch matches the requirements for a signal sequence for mitochondrial protein import. A mitochondrial location of the YDL202w gene product was proven by use of a carboxy terminally HA-tagged version. These findings clearly indicate that YDL202w encodes this mitochondrial ribosomal protein (YmL11).

Trans-activation of group II intron splicing by nuclear U5 snRNA.

Similarities between RNA splicing during autocatalytic excision of group II introns and pre-mRNA processing led to the hypothesis that group II introns might be the evolutionary predecessors of spliceosomal small nuclear RNAs. The ID3 subdomain stem-loop structure of group II introns, the proposed analogue of the spliceosomal U5 snRNA, is thought to be essential for 5' splice site recognition and anchoring of the free 5' exon. Using the group II intron bI1 we have analysed the role of ID3 in splicing. In the absence of ID3 the 5' splice site was recognized accurately and efficiently, but exon anchoring was greatly reduced. This step was restored in the presence of RNA fragments consisting of either the terminal stem-loop structure of ID3 or spliceosomal U5 snRNA. This suggests that the predominant role of both RNAs is to anchor the 5' exon during exon ligation. Furthermore, as U5 complements for the loss of ID3, a similar network of structural RNAs may form the catalytic core of both group II introns and spliceosomes.

Amino- and carboxy-terminal domains of the yeast Rab escort protein are both required for binding of Ypt small G proteins.

The Rab escort protein (REP) is an essential component of the heterotrimeric enzyme Rab geranylgeranyl transferase that modifies the carboxy-terminal cysteines of the Ras-like small G proteins belonging to the Rab/Ypt family. Deletions in the human CHM locus, encoding one of the two REPs known in humans, result in a retinal degenerative syndrome called choroideremia. The only known yeast homologue of the choroideremia gene product is encoded by an essential gene called MRS6. Besides three structurally conserved regions (SCRs) previously detected in the amino-terminal half of REPs and RabGDIs, three other regions in the carboxy-terminal domain (RCR 1-3) are here identified as being characteristic of REPs alone. We have performed the first mutational analysis of a REP protein to experimentally define the regions functionally important for Rab/Ypt protein binding, making use of the genetic system of the yeast Saccharomyces cerevisiae. This analysis has shown that the SCRs are necessary but not sufficient for Ypt1p binding by the yeast REP, the carboxy-terminal region also being required.

Mrs5p, an essential protein of the mitochondrial intermembrane space, affects protein import into yeast mitochondria.

We have isolated a yeast nuclear gene that suppresses the previously described respiration-deficient mrs2-1 mutation when present on a multicopy plasmid. Elevated gene dosage of this new gene, termed MRS5, suppresses also the pet phenotype of a mitochondrial splicing-deficient group II intron mutation M1301. The MRS5 gene product, a 13-kDa protein of low abundance, shows no similarity to other known proteins and is associated with the inner mitochondrial membrane, protruding into the intermembrane space. MRS5 codes for an essential protein, as the disruption of this gene is lethal even during growth on fermentable carbon sources. Thus, the Mrs5 protein seems to be involved in mitochondrial key functions aside from oxidative energy conservation, which is dispensable in fermenting yeast cells. Depletion of Mrs5p in yeast cells causes accumulation of unprocessed precursors of the mitochondrial hsp60 protein and defects in all cytochrome complexes. These findings suggest an essential role of Mrs5p in mitochondrial biogenesis.

Molecular characterization of the PEL1 gene encoding a putative phosphatidylserine synthase.

In the yeast Saccharomyces cerevisiae the PEL1 gene is essential for the viability of rho-/rhoo petite mutants, and its mutation in respiring cells results in a pleiotropic phenotype. Results of complementation analysis with different subclones of chromosomal DNA and re-sequencing of the YCL4w-YCL3w segment of chromsome III demonstrate that the coding region of the PEL1 gene corresponds to 1467 bp. The size of the PEL1 transcript in Northern blot analysis was estimated to be approximately 1.5 kb. Transcription initiation in wild-type cells was found to occur at the position -9 relative to the ATG. The PEL1 gene was moderately expressed irrespective of the state of the mitochondrial genome and the nature of the carbon sources. Disruption of the PEL1 gene was not lethal and resulted in the same phenotype as observed with the pel1 mutant, i.e. the cells were not able to survive ethidium bromide mutagenesis, were thermosensitive for growth on glucose at 37 degrees C and failed to grow on minimal glycerol medium. Although the Pel1 protein exhibits significant similarity to a family of phosphatidylserine synthases, the disrupted PEL1 gene was not complemented by the multicopy plasmid-borne CHO1 gene encoding an essential yeast phosphatidylserine synthase.

Overexpression of a novel member of the mitochondrial carrier family rescues defects in both DNA and RNA metabolism in yeast mitochondria.

The PIF1 and MRS2 gene products have previously been shown to be essential for mitochondrial DNA maintenance at elevated temperatures and mitochondrial group II intron splicing, respectively, in the yeast Saccharomyces cerevisiae. A multicopy suppressor capable of rescuing the respiratory deficient phenotype associated with null alleles of either gene has been isolated. This suppressor is a nuclear gene that was called RIM2/MRS12. The RIM2/MRS12 gene encodes a predicted protein of 377 amino acids that is essential for mitochondrial DNA metabolism and proper cell growth. Inactivation of this gene causes the total loss of mitochondrial DNA and, compared to wild-type rhoo controls, a slow-growth phenotype on media containing glucose. Analysis of the RIM2/MRS12 protein sequence suggests that RIM2/MRS12 encodes a novel member of the mitochondrial carrier family. In particular, a typical triplicate structure, where each repeat consists of two putative transmembrane segments separated by a hydrophilic loop, can be deduced from amino acid sequence comparisons and the hydropathy profile of RIM2/MRS12. Antibodies directed against the aminoterminus of RIM2/MRS12 detect this protein in mitochondria. The function of the RIM2/MRS12 protein and the substrates it might transport are discussed.

Mrs6p, the yeast homologue of the mammalian choroideraemia protein: immunological evidence for its function as the Ypt1p Rab escort protein.

The Saccharomyces cerevisiae MRS6 gene belongs to the same gene family as that responsible for the mammalian Rab escort protein (REP) and the Rab GDP dissociation inhibitor protein (GDI). Both REP and GDI are regulators of the Ras-related small G-proteins Rab/YPT1 which are involved in intracellular vesicular trafficking in yeast and in mammals. Here we characterize an antiserum directed against Mrs6p and show that it specifically inhibits the geranylation of the YPT1 protein in an in vitro assay. These findings provide direct evidence for the role of Mrs6p as the REP component of the yeast Rab geranylgeranyl transferase enzyme.