Structural and functional characterization of the N-terminal domain of the yeast Mg2+ channel Mrs2.

Mg(2+) translocation across cellular membranes is crucial for a myriad of physiological processes. Eukaryotic Mrs2 transporters are distantly related to the major bacterial Mg(2+) transporter CorA, the structure of which displays a bundle of giant α-helices forming a long pore that extends beyond the membrane before widening into a funnel-shaped cytosolic domain. Here, a functional and structural analysis of the regulatory domain of the eukaryotic Mg(2+) channel Mrs2 from the yeast inner mitochondrial membrane is presented using crystallography, genetics, biochemistry and fluorescence spectroscopy. Surprisingly, the fold of the Mrs2 regulatory domain bears notable differences compared with the related bacterial channel CorA. Nevertheless, structural analysis showed that analogous residues form functionally critical sites, notably the hydrophobic gate and the Mg(2+)-sensing site. Validation of candidate residues was performed by functional studies of mutants in isolated yeast mitochondria. Measurements of the Mg(2+) influx into mitochondria confirmed the involvement of Met309 as the major gating residue in Mrs2, corresponding to Met291 in CorA.

The mitochondrial carrier Rim2 co-imports pyrimidine nucleotides and iron.

Mitochondrial iron uptake is of key importance both for organelle function and cellular iron homoeostasis. The mitochondrial carrier family members Mrs3 and Mrs4 (homologues of vertebrate mitoferrin) function in organellar iron supply, yet other low efficiency transporters may exist. In Saccharomyces cerevisiae, overexpression of RIM2 (MRS12) encoding a mitochondrial pyrimidine nucleotide transporter can overcome the iron-related phenotypes of strains lacking both MRS3 and MRS4. In the present study we show by in vitro transport studies that Rim2 mediates the transport of iron and other divalent metal ions across the mitochondrial inner membrane in a pyrimidine nucleotide-dependent fashion. Mutations in the proposed substrate-binding site of Rim2 prevent both pyrimidine nucleotide and divalent ion transport. These results document that Rim2 catalyses the co-import of pyrimidine nucleotides and divalent metal ions including ferrous iron. The deletion of RIM2 alone has no significant effect on mitochondrial iron supply, Fe-S protein maturation and haem synthesis. However, RIM2 deletion in mrs3/4Δ cells aggravates their Fe-S protein maturation defect. We conclude that under normal physiological conditions Rim2 does not play a significant role in mitochondrial iron acquisition, yet, in the absence of the main iron transporters Mrs3 and Mrs4, this carrier can supply the mitochondrial matrix with iron in a pyrimidine-nucleotide-dependent fashion.

The G-M-N motif determines ion selectivity in the yeast magnesium channel Mrs2p.

The highly conserved G-M-N motif of the CorA-Mrs2-Alr1 family of Mg(2+) channels has been shown to be essential for Mg(2+) transport. We performed random mutagenesis of the G-M-N sequence of Saccharomyces cerevisiae Mrs2p in an unbiased genetic screen. A large number of mutants still capable of Mg(2+) influx, albeit below the wild-type level, were generated. Growth complementation assays, performed in media supplemented with Ca(2+) or Co(2+) or Mn(2+) or Zn(2+) at varying concentrations, lead to identification of mutants with reduced growth in the presence of Mn(2+) and Zn(2+). We hereby conclude that (1) at least two, but predominantly all three amino acids of the G-M-N motif must be replaced by certain combinations of other amino acids to remain functional, (2) replacement of any single amino acid within the G-M-N motif always impairs the function of Mrs2p, and (3) we show that the G-M-N motif determines ion selectivity, likely in concurrence with the negatively charged loop at the entrance of the channel thereby forming the Mrs2p selectivity filter.

Pun1p is a metal ion-inducible, calcineurin/Crz1p-regulated plasma membrane protein required for cell wall integrity.

Under conditions of environmental stress, the plasma membrane is involved in several regulatory processes to promote cell survival, like maintenance of signaling pathways, cell wall organization and intracellular ion homeostasis. PUN1 encodes a plasma membrane protein localizing to the ergosterol-rich membrane compartment occupied also by the arginine permease Can1. We found that the PUN1 (YLR414c) gene is transcriptionally induced upon metal ion stress. Northern blot analysis of the transcriptional regulation of PUN1 showed that the calcium dependent transcription factor Crz1p is required for PUN1 induction upon heavy metal stress. Here we report that mutants deleted for PUN1 exhibit increased metal ion sensitivity and morphological abnormalities. Microscopical and ultrastructural observations revealed a severe cell wall defect of pun1∆ mutants. By using chemical cross-linking, Blue native electrophoresis, and co-immunoprecipitation we found that Pun1p forms homo-oligomeric protein complexes. We propose that Pun1p is a stress-regulated factor required for cell wall integrity, thereby expanding the functional significance of lateral plasma membrane compartments.

Adjuvating the adjuvant: facilitated delivery of an immunomodulatory oligonucleotide to TLR9 by a cationic antimicrobial peptide in dendritic cells.

IC31(®) is a novel bi-component vaccine adjuvant consisting of the peptide KLKL(5)KLK (KLK) and the TLR9 agonist oligonucleotide d(IC)(13) (ODN1a). While membrane-interacting properties of KLK and immuno-modulating capabilities of ODN1a have been characterized in detail, little is known of how these two molecules function together and synergize in interacting with their primary target cells, dendritic cells (DCs). We have found that KLK-triggered aggregates entrapped ODN1a and these complexes readily associated with the DC cell surface. KLK stimulated the uptake and internalization of ODN1a via endocytosis, while the bulk of the peptide remained associated with the cell periphery. ODN1a co-localized with early and late endosomes as well as endoplasmic reticular structures. ODN1a co-localized with TLR9 positive compartments following KLK mediated uptake. These features did not depend on the expression of TLR-9. Our results reveal novel mechanisms that allow KLK to enhance the effects of the TLR-9 ligand ODN1a in immunomodulation.

Functional analysis of the conserved hydrophobic gate region of the magnesium transporter CorA.

The Leu294 residue in the cytoplasmic neck of Thermotoga maritima CorA is considered to be the main gate for Mg2+ transport. We created three site-directed mutants at this position: in the Leu294Asp and Leu294Gly mutants we observed a defect in closing of the pore, while in the Leu294Arg mutant not only gating, but also the regulation of Mg2+ uptake was affected. Our results confirmed the importance of the Leu294 for gating of Mg2+ transport and in addition revealed the influence of the charge and structural features of the amino acid residues on the gating mechanism.

Lpe10p modulates the activity of the Mrs2p-based yeast mitochondrial Mg2+ channel.

Saccharomyces cerevisiae Lpe10p is a homologue of the Mg(2+)-channel-forming protein Mrs2p in the inner mitochondrial membrane. Deletion of MRS2, LPE10 or both results in a petite phenotype, which exhibits a respiratory growth defect on nonfermentable carbon sources. Only coexpression of MRS2 and LPE10 leads to full complementation of the mrs2Delta/lpe10Delta double disruption, indicating that these two proteins cannot substitute for each other. Here, we show that deletion of LPE10 results in a loss of rapid Mg(2+) influx into mitochondria, as has been reported for MRS2 deletion. Additionally, we found a considerable loss of the mitochondrial membrane potential (DeltaPsi) in the absence of Lpe10p, which was not detected in mrs2Delta cells. Addition of the K(+)/H(+)-exchanger nigericin, which artificially increases DeltaPsi, led to restoration of Mg(2+) influx into mitochondria in lpe10Delta cells, but not in mrs2Delta/lpe10Delta cells. Mutational analysis of Lpe10p and domain swaps between Mrs2p and Lpe10p suggested that the maintenance of DeltaPsi and that of Mg(2+) influx are functionally separated. Cross-linking and Blue native PAGE experiments indicated interaction of Lpe10p with the Mrs2p-containing channel complex. Using the patch clamp technique, we showed that Lpe10p was not able to mediate high-capacity Mg(2+) influx into mitochondrial inner membrane vesicles without the presence of Mrs2p. Instead, coexpression of Lpe10p and Mrs2p yielded a unique, reduced conductance in comparison to that of Mrs2p channels. In summary, the data presented show that the interplay of Lpe10p and Mrs2p is of central significance for the transport of Mg(2+) into mitochondria of S. cerevisiae.

Crystallization and preliminary X-ray diffraction analysis of the N-terminal domain of Mrs2, a magnesium ion transporter from yeast inner mitochondrial membrane.

Mrs2 transporters are distantly related to the major bacterial Mg(2+) transporter CorA and to Alr1, which is found in the plasma membranes of lower eukaryotes. Common features of all Mrs2 proteins are the presence of an N-terminal soluble domain followed by two adjacent transmembrane helices (TM1 and TM2) near the C-terminus and of the highly conserved F/Y-G-M-N sequence motif at the end of TM1. The inner mitochondrial domain of the Mrs2 from Saccharomyces cerevisae was overexpressed, purified and crystallized in two different crystal forms corresponding to an orthorhombic and a hexagonal space group. The crystals diffracted X-rays to 1.83 and 4.16 A resolution, respectively. Matthews volume calculations suggested the presence of one molecule per asymmetric unit in the orthorhombic crystal form and of five or six molecules per asymmetric unit in the hexagonal crystal form. The phase problem was solved for the orthorhombic form by a single-wavelength anomalous dispersion experiment exploiting the sulfur anomalous signal.

Novel components of an active mitochondrial K(+)/H(+) exchange.

Defects of the mitochondrial K(+)/H(+) exchanger (KHE) result in increased matrix K(+) content, swelling, and autophagic decay of the organelle. We have previously identified the yeast Mdm38 and its human homologue LETM1, the candidate gene for seizures in Wolf-Hirschhorn syndrome, as essential components of the KHE. In a genome-wide screen for multicopy suppressors of the pet(-) (reduced growth on nonfermentable substrate) phenotype of mdm38Delta mutants, we now characterized the mitochondrial carriers PIC2 and MRS3 as moderate suppressors and MRS7 and YDL183c as strong suppressors. Like Mdm38p, Mrs7p and Ydl183cp are mitochondrial inner membrane proteins and constituents of approximately 500-kDa protein complexes. Triple mutant strains (mdm38Delta mrs7Delta ydl183cDelta) exhibit a remarkably stronger pet(-) phenotype than mdm38Delta and a general growth reduction. They totally lack KHE activity, show a dramatic drop of mitochondrial membrane potential, and heavy fragmentation of mitochondria and vacuoles. Nigericin, an ionophore with KHE activity, fully restores growth of the triple mutant, indicating that loss of KHE activity is the underlying cause of its phenotype. Mdm38p or overexpression of Mrs7p, Ydl183cp, or LETM1 in the triple mutant rescues growth and KHE activity. A LETM1 human homologue, HCCR-1/LETMD1, described as an oncogene, partially suppresses the yeast triple mutant phenotype. Based on these results, we propose that Ydl183p and the Mdm38p homologues Mrs7p, LETM1, and HCCR-1 are involved in the formation of an active KHE system.

A Drosophila mutant of LETM1, a candidate gene for seizures in Wolf-Hirschhorn syndrome.

Human Wolf-Hirschhorn syndrome (WHS) is a multigenic disorder resulting from a hemizygous deletion on chromosome 4. LETM1 is the best candidate gene for seizures, the strongest haploinsufficiency phenotype of WHS patients. Here, we identify the Drosophila gene CG4589 as the ortholog of LETM1 and name the gene DmLETM1. Using RNA interference approaches in both Drosophila melanogaster cultured cells and the adult fly, we have assayed the effects of down-regulating the LETM1 gene on mitochondrial function. We also show that DmLETM1 complements growth and mitochondrial K(+)/H(+) exchange (KHE) activity in yeast deficient for LETM1. Genetic studies allowing the conditional inactivation of LETM1 function in specific tissues demonstrate that the depletion of DmLETM1 results in roughening of the adult eye, mitochondrial swelling and developmental lethality in third-instar larvae, possibly the result of deregulated mitophagy. Neuronal specific down-regulation of DmLETM1 results in impairment of locomotor behavior in the fly and reduced synaptic neurotransmitter release. Taken together our results demonstrate the function of DmLETM1 as a mitochondrial osmoregulator through its KHE activity and uncover a pathophysiological WHS phenotype in the model organism D. melanogaster.

A root-expressed magnesium transporter of the MRS2/MGT gene family in Arabidopsis thaliana allows for growth in low-Mg2+ environments.

The MRS2/MGT gene family in Arabidopsis thaliana belongs to the superfamily of CorA-MRS2-ALR-type membrane proteins. Proteins of this type are characterized by a GMN tripeptide motif (Gly-Met-Asn) at the end of the first of two C-terminal transmembrane domains and have been characterized as magnesium transporters. Using the recently established mag-fura-2 system allowing direct measurement of Mg(2+) uptake into mitochondria of Saccharomyces cerevisiae, we find that all members of the Arabidopsis family complement the corresponding yeast mrs2 mutant. Highly different patterns of tissue-specific expression were observed for the MRS2/MGT family members in planta. Six of them are expressed in root tissues, indicating a possible involvement in plant magnesium supply and distribution after uptake from the soil substrate. Homozygous T-DNA insertion knockout lines were obtained for four members of the MRS2/MGT gene family. A strong, magnesium-dependent phenotype of growth retardation was found for mrs2-7 when Mg(2+) concentrations were lowered to 50 microM in hydroponic cultures. Ectopic overexpression of MRS2-7 from the cauliflower mosaic virus 35S promoter results in complementation and increased biomass accumulation. Green fluorescent protein reporter gene fusions indicate a location of MRS2-7 in the endomembrane system. Hence, contrary to what is frequently found in analyses of plant gene families, a single gene family member knockout results in a strong, environmentally dependent phenotype.

Determination of yeast mitochondrial KHE activity, osmotic swelling and mitophagy.

The mitochondrial K(+)/H(+) exchanger (KHE) is a key regulator of mitochondrial K(+), the most abundant cellular cation, and thus for volume control of the organelle. Downregulation of the mitochondrial KHE results in osmotic swelling and autophagic degradation of the organelle. This chapter describes methods to shut-off expression of Mdm38p, an essential factor of the mitochondrial KHE, and to observe the cellular consequences thereof, in particular changes in KHE activity and morphogenetic changes of mitochondria by applying new techniques developed in our laboratories.

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane.

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe(2+) homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe(2+) utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe(2+) which was driven by the external Fe(2+) concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Delta failed to show this rapid Fe(2+) uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe(2+) uptake rates. Cu(2+) was transported at similar rates as Fe(2+), while other divalent cations, such as Zn(2+) and Cd(2+) apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe(2+) across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.

Pathophysiology of mitochondrial volume homeostasis: potassium transport and permeability transition.

Regulation of mitochondrial volume is a key issue in cellular pathophysiology. Mitochondrial volume and shape changes can occur following regulated fission-fusion events, which are modulated by a complex network of cytosolic and mitochondrial proteins; and through regulation of ion transport across the inner membrane. In this review we will cover mitochondrial volume homeostasis that depends on (i) monovalent cation transport across the inner membrane, a regulated process that couples electrophoretic K(+) influx on K(+) channels to K(+) extrusion through the K(+)-H(+) exchanger; (ii) the permeability transition, a loss of inner membrane permeability that may be instrumental in triggering cell death. Specific emphasis will be placed on molecular advances on the nature of the transport protein(s) involved, and/or on diseases that depend on mitochondrial volume dysregulation.

Conditional knockdown of hMRS2 results in loss of mitochondrial Mg(2+) uptake and cell death.

The human gene MRS2L encodes a mitochondrial protein distantly related to CorA Mg(2+) transport proteins. Constitutive shRNA-mediated knockdown of hMRS2 in human HEK-293 cell line was found here to cause death. To further study its role in Mg(2+) transport, we have established stable cell lines with conditionally expressing shRNAs directed against hMRS2L. The cells expressing shRNA for several generations exhibited lower steady-state levels of free mitochondrial Mg(2+) ([Mg(2+)](m)) and reduced capacity of mitochondrial Mg(2+) uptake than control cells. Long-term expression of shRNAs resulted in loss of mitochondrial respiratory complex I, decreased mitochondrial membrane potential and cell death. We conclude that hMrs2 is the major transport protein for Mg (+) uptake into mitochondria and that expression of hMrs2 is essential for the maintenance of respiratory complex I and cell viability.

Arsenic toxicity to Saccharomyces cerevisiae is a consequence of inhibition of the TORC1 kinase combined with a chronic stress response.

The conserved Target Of Rapamycin (TOR) growth control signaling pathway is a major regulator of genes required for protein synthesis. The ubiquitous toxic metalloid arsenic, as well as mercury and nickel, are shown here to efficiently inhibit the rapamycin-sensitive TORC1 (TOR complex 1) protein kinase. This rapid inhibition of the TORC1 kinase is demonstrated in vivo by the dephosphorylation and inactivation of its downstream effector, the yeast S6 kinase homolog Sch9. Arsenic, mercury, and nickel cause reduction of transcription of ribosome biogenesis genes, which are under the control of Sfp1, a TORC1-regulated transcriptional activator. We report that arsenic stress deactivates Sfp1 as it becomes dephosphorylated, dissociates from chromatin, and exits the nucleus. Curiously, whereas loss of SFP1 function leads to increased arsenic resistance, absence of TOR1 or SCH9 has the opposite effect suggesting that TORC1 has a role beyond down-regulation of Sfp1. Indeed, we show that arsenic activates the transcription factors Msn2 and Msn4 both of which are targets of TORC1 and protein kinase A (PKA). In contrast to TORC1, PKA activity is not repressed during acute arsenic stress. A normal level of PKA activity might serve to dampen the stress response since hyperactive Msn2 will decrease arsenic tolerance. Thus arsenic toxicity in yeast might be determined by the balance between chronic activation of general stress factors in combination with lowered TORC1 kinase activity.

Unique membrane-interacting properties of the immunostimulatory cationic peptide KLKL(5)KLK (KLK).

We have monitored the effects of KLKL(5)KLK (KLK), a derivative of a natural cationic antimicrobial peptide (CAP) on isolated membrane vesicles, and investigated the partition of the peptide within these structures. KLK readily interacted with fluorescent dyes entrapped in the vesicles without apparent pore formation. Fractionation of vesicles revealed KLK predominantly in the membrane. Peptide-treated vesicles appeared with generally disorganized bilayers. While KLK showed no effect on osmotic resistance of human erythrocytes, dramatic decrease in core and surface membrane fluidity was observed in peptide-treated erythrocyte ghosts as measured by fluorescence anisotropy. Finally, CD spectroscopy revealed lipid-induced random coil to beta-sheet and beta-sheet to alpha-helix conformational transitions of KLK. Together with the oligonucleotide oligo-d(IC)(13) [ODN1a], KLK functions as a novel adjuvant, termed IC31. Among other immunological effects, KLK appears to facilitate the uptake and delivery of ODN1a into cellular compartments, but the nature of KLK's interaction with the cell surface and other membrane-bordered compartments remains unknown. Our results suggest a profound membrane interacting property of KLK that might contribute to the immunostimulatory activities of IC31.

Mrs2p forms a high conductance Mg2+ selective channel in mitochondria.

Members of the CorA-Mrs2-Alr1 superfamily of Mg(2+) transporters are ubiquitous among pro- and eukaryotes. The crystal structure of a bacterial CorA protein has recently been solved, but the mode of ion transport of this protein family remained obscure. Using single channel patch clamping we unequivocally show here that the mitochondrial Mrs2 protein forms a Mg(2+)-selective channel of high conductance (155 pS). It has an open probability of approximately 60% in the absence of Mg(2+) at the matrix site, which decreases to approximately 20% in its presence. With a lower conductance ( approximately 45 pS) the Mrs2 channel is also permeable for Ni(2+), whereas no permeability has been observed for either Ca(2+), Mn(2+), or Co(2+). Mutational changes in key domains of Mrs2p are shown either to abolish its Mg(2+) transport or to change its characteristics toward more open and partly deregulated states. We conclude that Mrs2p forms a high conductance Mg(2+) selective channel that controls Mg(2+) influx into mitochondria by an intrinsic negative feedback mechanism.

Mg2+ deprivation elicits rapid Ca2+ uptake and activates Ca2+/calcineurin signaling in Saccharomyces cerevisiae.

To learn about the cellular processes involved in Mg(2+) homeostasis and the mechanisms allowing cells to cope with low Mg(2+) availability, we performed RNA expression-profiling experiments and followed changes in gene activity upon Mg(2+) depletion on a genome-wide scale. A striking portion of genes up-regulated under Mg(2+) depletion are also induced by high Ca(2+) and/or alkalinization. Among the genes significantly up-regulated by Mg(2+) starvation, Ca(2+) stress, and alkalinization are ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter). We show that up-regulation of these genes is dependent on the calcineurin/Crz1p (calcineurin-responsive zinc finger protein) signaling pathway. Similarly to Ca(2+) stress, Mg(2+) starvation induces translocation of the transcription factor Crz1p from the cytoplasm into the nucleus. The up-regulation of ENA1 and PHO89 upon Mg(2+) starvation depends on extracellular Ca(2+). Using fluorescence resonance energy transfer microscopy, we demonstrate that removal of Mg(2+) results in an immediate increase in free cytoplasmic Ca(2+). This effect is dependent on external Ca(2+). The results presented indicate that Mg(2+) depletion in yeast cells leads to enhanced cellular Ca(2+) concentrations, which activate the Crz1p/calcineurin pathway. We provide evidence that calcineurin/Crz1p signaling is crucial for yeast cells to cope with Mg(2+) depletion stress.

Oligomerization of the Mg2+-transport proteins Alr1p and Alr2p in yeast plasma membrane.

Alr1p is an integral plasma membrane protein essential for uptake of Mg(2+) into yeast cells. Homologs of Alr1p are restricted to fungi and some protozoa. Alr1-type proteins are distant relatives of the mitochondrial and bacterial Mg(2+)-transport proteins, Mrs2p and CorA, respectively, with which they have two adjacent TM domains and a short Mg(2+) signature motif in common. The yeast genome encodes a close homolog of Alr1p, named Alr2p. Both proteins are shown here to be present in the plasma membrane. Alr2p contributes poorly to Mg(2+) uptake. Substitution of a single arginine with a glutamic acid residue in the loop connecting the two TM domains at the cell surface greatly improves its function. Both proteins are shown to form homo-oligomers as well as hetero-oligomers. Wild-type Alr2p and mutant Alr1 proteins can have dominant-negative effects on wild-type Alr1p activity, presumably through oligomerization of low-function with full-function proteins. Chemical cross-linking indicates the presence of Alr1 oligomers, and split-ubiquitin assays reveal Alr1p-Alr1p, Alr2p-Alr2p, and Alr1p-Alr2p interactions. These assays also show that both the N-terminus and C-terminus of Alr1p and Alr2p are exposed to the inner side of the plasma membrane.