The Channel-Forming Sym1 Protein Is Transported by the TIM23 Complex in a Presequence-Independent Manner

The majority of multispanning inner mitochondrial membrane proteins utilize internal targeting signals, which direct them to the carrier translocase (TIM22 complex), for their import. MPV17 and its Saccharomyces cerevisiae orthologue Sym1 are multispanning inner membrane proteins of unknown function with an amino-terminal presequence that suggests they may be targeted to the mitochondria. Mutations affecting MPV17 are associated with mitochondrial DNA depletion syndrome (MDDS). Reconstitution of purified Sym1 into planar lipid bilayers and electrophysiological measurements have demonstrated that Sym1 forms a membrane pore. To address the biogenesis of Sym1, which oligomerizes in the inner mitochondrial membrane, we studied its import and assembly pathway. Sym1 forms a transport intermediate at the translocase of the outer membrane (TOM) complex. Surprisingly, Sym1 was not transported into mitochondria by an amino-terminal signal, and in contrast to what has been observed in carrier proteins, Sym1 transport and assembly into the inner membrane were independent of small translocase of mitochondrial inner membrane (TIM) and TIM22 complexes. Instead, Sym1 required the presequence of translocase for its biogenesis. Our analyses have revealed a novel transport mechanism for a polytopic membrane protein in which internal signals direct the precursor into the inner membrane via the TIM23 complex, indicating a presequence-independent function of this translocase.     

A novel alternative spliced Mpv17-like protein isoform localizes in cytosol and is expressed in a kidney- and adult-specific manner

Mpv17-like protein (M-LP) has been identified as a new protein that shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. We previously showed that the originally identified M-LP isoform, designated M-LPL, is, like Mpv17, localized in peroxisomes, and that transfection with M-LPL up-regulates expression of the manganese superoxide dismutase (SOD2) gene [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene. J. Biol. Chem. 278 (2003) 6301-6306.]. We report here the identification of a novel alternative splicing product of the M-LP gene, designated M-LPS. A comparison of the genomic sequence with the cDNA sequences and an analysis of 5'-flanking regions revealed that the two isoforms are generated by alternative usage of two promoters. M-LPS consists of the C-terminal half of M-LPL (90 amino acids) and therefore lacks the peroxisome targeting signal of membrane protein that exists near the N-terminus of M-LPL. Expression of green fluorescent protein-tagged M-LPS in COS-7 cells demonstrated that M-LPS localizes in the cytosol. In mice, M-LPS is expressed exclusively in kidneys after the age of 6 weeks. Moreover, quantitative real-time PCR analysis revealed that transfection with M-LPS up-regulates expression of the SOD2 gene and down-regulates expression of the cellular glutathione peroxidase (Gpx1) and plasma glutathione peroxidase (Gpx3) genes. Taken together, these results suggest different functional attributes of the two M-LP isoforms during aging and development.     

The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species.

The mutant mouse strain Mpv17 carries a retroviral insert in its genome which inactivates the Mpv17 gene. At a young age these mice develop glomerulosclerosis and nephrotic syndrome which resembles human disease. We show here that the Mpv17 gene product is highly conserved and encodes a peroxisomal protein. Loss of the Mpv17 protein does not impair peroxisome biogenesis but instead leads to a reduced ability to produce reactive oxygen species (ROS). In turn, overproduction of the Mpv17 gene in transfected cells results in dramatically enhanced levels of intracellular ROS indicating a direct involvement of Mpv17 in ROS production. These data reveal a role for the Mpv17 protein in peroxisomal reactive oxygen metabolism and establish a novel link between peroxisomal ROS production and glomerulosclerosis.     

Human Mpv17-like protein is localized in peroxisomes and regulates expression of antioxidant enzymes

M-LP (Mpv17-like protein) is a protein that was initially identified in mouse tissues and shows high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early-onset glomerulosclerosis [R. Iida, T. Yasuda, E. Tsubota, H. Takatsuka, M. Masuyama, T. Matsuki, K. Kishi, M-LP, Mpv17-like protein, has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene, J. Biol. Chem. 278 (2003) 6301-6306]. Here we report the identification and characterization of a human homolog of the M-LP (M-LPH) gene. The M-LPH gene is composed of four exons, extends over 14kb on chromosome 16p13.1, and is expressed as two alternatively spliced variants comprising four and three exons, respectively, which include open-reading frames encoding two distinct isoforms composed of 196 (M-LPH1) and 147 (M-LPH2) amino acids, respectively. These two variants were expressed ubiquitously in human tissues, however only M-LPH1 was detected at the protein level. Dual-color confocal analysis of COS-7 cells transfected with a green fluorescent protein-tagged M-LPH1 demonstrated that M-LPH1 is localized in peroxisomes. In order to elucidate the function of M-LPH1, we examined the mRNA levels of several enzymes involved in the metabolism of reactive oxygen species in COS-7 cells and found that transfection with M-LPH1 down-regulates expression of the plasma glutathione peroxidase and catalase genes. These results show the existence of the human homolog of M-LP and its participation in reactive oxygen species metabolism.     

M-LP,Mpv17-like protein,has a peroxisomal membrane targeting signal comprising a transmembrane domain and a positively charged loop and up-regulates expression of the manganese superoxide dismutase gene

M-LP (Mpv17-like protein) has been identified as a new protein that has high sequence homology with Mpv17 protein, a peroxisomal membrane protein involved in the development of early onset glomerulosclerosis. In this study, we verified the peroxisomal localization of M-LP by performing dual-color confocal analysis of COS-7 cells cotransfected with green fluorescent protein-tagged M-LP and DsRED2-PTS1, a red fluorescent peroxisomal marker. To characterize the peroxisomal membrane targeting signal, we examined the intracellular localizations of several green fluorescent protein-tagged deletion mutants and demonstrated that, of the three transmembrane segments predicted, the first near the NH(2) terminus and NH(2)-terminal half of the following loop region, which is abundant in positively charged amino acids, were necessary and sufficient for peroxisomal targeting. To elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species metabolism in COS-7 cells and found that transfection with M-LP increased the superoxide dismutase activity significantly. Quantitative real-time PCR analysis revealed that the manganese SOD (SOD2) mRNA level of COS-7 cells transfected with M-LP was elevated. These results indicate that M-LP participates in reactive oxygen species metabolism.     

Heat shock response of the archaebacterium Methanococcus voltae.

The general properties of the heat shock response of the archaebacterium Methanococcus voltae were characterized. The induction of 11 heat shock proteins, with apparent molecular weights ranging from 18,000 to 90,000, occurred optimally at 40 to 50 degrees C. Some of the heat shock proteins were preferentially enriched in either the soluble (cytoplasm) or particulate (membrane) fraction. Alternative stresses (ethanol, hydrogen peroxide, NaCl) stimulated the synthesis of subsets of the heat shock proteins as well as unique proteins. Western blot (immunoblot) analysis, in which antisera to Escherichia coli heat shock proteins (DnaK and GroEL) were used, did not detect any immunologically cross-reactive proteins. In addition, Southern blot analysis did not reveal any homology between M. voltae and four highly conserved heat shock genes, mopB and dnaK from E. coli and hsp70 genes from Drosophila species and Saccharomyces cerevisiae.     

Stress tolerance in a yeast lipid mutant: membrane lipids influence tolerance to heat and ethanol independently of heat shock proteins and trehalose.

The response of a yeast unsaturated fatty acid auxotroph, defective in delta 9-desaturase activity, to heat and ethanol stresses was examined. The most heat- and ethanol-tolerant cells had membranes enriched with oleic acid (C18:1), followed in order by cells enriched with linoleic (C18:2) and linolenic (C18:3) acids. Cells subjected to a heat shock (25-37 degrees C for 30 min) accumulated trehalose and synthesized typical heat shock proteins. Although there were no obvious differences in protein profiles attributable to lipid supplementation of the mutant, relative protein synthesis as determined by densitometric analysis of autoradiograms suggested that hsp expression was different. However, there was no consistent relationship between the synthesis of heat shock proteins and the acquisition of thermotolerance in the lipid supplemented auxotroph or related wild type. Furthermore, trehalose accumulation was also not closely related to stress tolerance. On the other hand, the data presented indicated a more consistent role for membrane lipid composition in stress tolerance than trehalose, heat shock proteins, or ergosterol. We suggest that the sensitivity of C18:3-enriched cells to heat and ethanol may be attributable to membrane damage associated with increases in membrane fluidity and oxygen-derived free radical attack of membrane lipids.     

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ?C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1? cells grown on synthetic media at both 30 ?C and 37 ?C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1?gpd1? double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.     

Abnormal basement membrane in the inner ear and the kidney of the Mpv17-/- mouse strain: ultrastructural and immunohistochemical investigations

The loss of the function of the peroxisomal Mpv17-protein and associated imbalanced radical oxygen species (ROS) homeostasis leads to an early onset of focal segmental glomerulosclerosis and sensorineural deafness associated with severe degeneration of cochlear structures. An excessive enlargement of basal laminae of the stria vascularis capillaries and glomeruli indicates numerous changes in their molecular composition. The basement membrane (BM) of the glomeruli and the stria vascularis are simultaneously affected in early stages of the disease and the lamination, splitting of the membrane and formation of the “basket weaving” seen at the onset of the disease in the kidney are similar to the ultrastructural alterations characteristic for Alportȁ9s syndrome. The progressive alteration of the BMs is accompanied by irregularity in the distribution of the collagen IV subunits and by an accumulation of the laminin B2(γ1) in the inner ear and B(β1) in the kidney. Since Mpv17 protein contributes to ROS homeostasis, further studies are necessary to elucidate downstream signaling molecules activated by ROS. These studies explain the cellular responses to missing Mpv17-protein, such as accumulation of the extracellular matrix, degeneration, and apoptosis in the inner ear.     

Heat-shock proteins in membrane vesicles of Bacillus subtilis

Fractionation of B. subtilis cells after heat shock, from 37 degrees C to 54 degrees C, shows an increase in synthesis of proteins localized in cell membranes and a decrease in synthesis of proteins localized in cytosol. There is no such effect of heat shock at temperature of 45 degrees C. Autoradiograms of electrophoretically separated proteins, labelled during heat shock at 54 degrees C, reveal 26 heat-shock proteins (hsps) in membrane vesicles and 11 hsps in cytosol, five of which are common to both fractions. Heat shock at 45 degrees C induces 18 hsps localized in membrane vesicles and 13 hsps localized in cytosol, six of which are common to both fractions. Results are interpreted as showing a relevant role of membrane proteins in cell response to shock at high temperature, pointing to two steps of defense against heat stress.     

Expression of the Recessive Glomerulosclerosis Gene Mpv17 Regulates MMP-2 Expression in Fibroblasts, the Kidney, and the Inner Ear of Mice

The recessive mouse mutant Mpv17 is characterized by the development of early-onset glomerulosclerosis, concomitant hypertension, and structural alterations of the inner ear. The primary cause of the disease is the loss of function of the Mpv17 protein, a peroxisomal gene product involved in reactive oxygen metabolism. In our search of a common mediator exerting effects on several aspects of the phenotype, we discovered that the absence of the Mpv17 gene product causes a strong increase in matrix metalloproteinase 2 (MMP-2) expression. This was seen in the kidney and cochlea of Mpv17-negative mice as well as in tissue culture cells derived from these animals. When these cells were transfected with the human Mpv17 homolog, an inverse causal relationship between Mpv17 and MMP-2 expression was established. These results indicate that the Mpv17 protein plays a crucial role in the regulation of MMP-2 and suggest that enhanced MMP-2 expression might mediate the mechanisms leading to glomerulosclerosis, inner ear disease, and hypertension in this model.     

The peroxin Pex17p of the yeast Yarrowia lipolytica is associated peripherally with the peroxisomal membrane and is required for the import of a subset of matrix proteins.

PEX genes encode peroxins, which are required for the biogenesis of peroxisomes. The Yarrowia lipolytica PEX17 gene encodes the peroxin Pex17p, which is 671 amino acids in length and has a predicted molecular mass of 75,588 Da. Pex17p is peripherally associated with the peroxisomal membrane. The carboxyl-terminal tripeptide, Gly-Thr-Leu, of Pex17p is not necessary for its targeting to peroxisomes. Synthesis of Pex17p is low in cells grown in glucose-containing medium and increases after the cells are shifted to oleic acid-containing medium. Cells of the pex17-1 mutant, the original mutant strain, and the pex17-KA mutant, a strain in which most of the PEX17 gene is deleted, fail to form normal peroxisomes but instead contain numerous large, multimembraned structures. The import of peroxisomal matrix proteins in these mutants is selectively impaired. This selective import is not a function of the nature of the peroxisomal targeting signal. We suggest a regulatory role for Pex17p in the import of a subset of matrix proteins into peroxisomes.     

Arabidopsis 22-kilodalton peroxisomal membrane protein. Nucleotide sequence analysis and biochemical characterization.

We sequenced and characterized PMP22 (22-kD peroxisomal membrane protein) from Arabidopsis, which shares 28% to 30% amino acid identity and 55% to 57% similarity to two related mammalian peroxisomal membrane proteins, PMP22 and Mpv17. Subcellular fractionation studies confirmed that the Arabidopsis PMP22 is a genuine peroxisomal membrane protein. Biochemical analyses established that the Arabidopsis PMP22 is an integral membrane protein that is completely embedded in the lipid bilayer. In vitro import assays demonstrated that the protein is inserted into the membrane posttranslationally in the absence of ATP, but that ATP stimulates the assembly into the native state. Arabidopsis PMP22 is expressed in all organs of the mature plant and in tissue-cultured cells. Expression of PMP22 is not associated with a specific peroxisome type, as it is detected in seeds and throughout postgerminative growth as cotyledon peroxisomes undergo conversion from glyoxysomes to leaf-type peroxisomes. Although PMP22 shows increased accumulation during the growth of young seedlings, its expression is not stimulated by light.     

Cloning and characterization of the CSF1 gene of Saccharomyces cerevisiae, which is required for nutrient uptake at low temperature

We have isolated cold-sensitive fermentation mutants (Csf mutants) of a commercial baker's yeast that have practically no fermentation capacity at 5 degrees C and return to their normal capacity at 25 to 40 degrees C. CSF1 was cloned by functional complementation of the Csf phenotype. CSF1 contain an open reading frame of 8,874 nucleotides, encoding a protein of 2,958 amino acids. The nucleotide sequence was identical to that of the YLR087C gene in the Saccharomyces genome database, but there was no information about the function of the predicted CSF1 (YLR087C) protein. Gene disruption shows that CSF1 is required for growth and fermentation only at low temperatures. Permeabilized cells of the disruptant showed nearly the same ethanol production rate as those of the parent strain, even at 10 degrees C. The disruptant cells had the same glucose uptake rates as the parental cells at 30 degrees C, but three- to fivefold-lower rates than the parental cells at 10 degrees C. These findings suggest that CSF1 associates with a new nutrient transport system which exists on the plasma membrane and is required only at low temperature.