Wolfram syndrome is an autosomal recessive disorder characterized by juvenile diabetes mellitus, diabetes insipidus, optic atrophy and a number of neurological symptoms including deafness, ataxia and peripheral neuropathy. Mitochondrial DNA deletions have been described in a few patients and a locus has been mapped to 4p16 by linkage analysis. Susceptibility to psychiatric illness is reported to be high in affected individuals and increased in heterozygous carriers in Wolfram syndrome families. We screened four candidate genes in a refined critical linkage interval covered by an unfinished genomic sequence of 600 kb. One of these genes, subsequently named wolframin, codes for a predicted transmembrane protein which was expressed in various tissues, including brain and pancreas, and carried loss-of-function mutations in both alleles in Wolfram syndrome patients.

Mutations of the WFS1 gene are responsible for Wolfram syndrome, a rare, recessive disorder characterized by early-onset, non-autoimmune diabetes mellitus, optic atrophy and further neurological and endocrinological abnormalities. The WFS1 gene encodes wolframin, a putative multispanning membrane glycoprotein of the endoplasmic reticulum. The function of wolframin is completely unknown. In order to characterize wolframin, we have generated polyclonal antibodies against both hydrophilic termini of the protein. Wolframin was found to be ubiquitously expressed with highest levels in brain, pancreas, heart and insulinoma beta-cell lines. Analysis of the structural features provides experimental evidence that wolframin contains nine transmembrane segments and is embedded in the membrane in an N(cyt)/C(lum) topology. Wolframin assembles into higher molecular weight complexes of approximately 400 kDa in the membrane. Pulse-chase experiments demonstrate that during maturation wolframin is N-glycosylated but lacks proteolytical processing. Moreover, N-glycosylation appears to be essential for the biogenesis and stability of wolframin. Here we investigate, for the first time, the molecular mechanisms that cause loss-of-function of wolframin in affected individuals. In patients harboring nonsense mutations complete absence of the mutated wolframin is caused by instability and rapid decay of WFS1 nonsense transcripts. In a patient carrying a compound heterozygous missense mutation, R629W, we found markedly reduced steady-state levels of wolframin. Pulse-chase experiments of mutant wolframin expressed in COS-7 cells indicated that the R629W mutation leads to instability and strongly reduced half-life of wolframin. Thus, the Wolfram syndrome in patients investigated here is caused by reduced protein dosage rather than dysfunction of the mutant wolframin.

Mitochondrial (mt)DNA haplogroups in a German control group (n = 67) were characterized by screening mitochondrial coding regions encompassing most of the ND, tRNA and cyt b genes. We used a PCR-SSCP screening approach followed by direct sequencing of polymorphic mtDNA fragments. Five major mtDNA lineages, diverging in at least nine different haplogroups, could be defined by characteristic polymorphic sites in mitochondrial genes. Additional sequencing of two hypervariable segments (HVS-I and II) of the non-coding displacement (D) loop in all control subjects revealed that certain D loop variants were strongly correlated with lineages and haplogroups, while others represented hotspots occurring frequently in different haplogroups. The existence of identified lineages and haplogroups received support from data in the literature, obtained by use of different approaches. Subsequently, we investigated four disease groups for association with these haplogroups: (i) LHON patients (n = 55) carrying at least one of the primary/intermediate LHON mutations at nt 3460, 11778, 14484 and/or 15257; (ii) patients suffering from Wolfram or DIDMOAD syndrome (n = 8); (iii) MELAS patients (n = 9); (iv) a group of children, who died from 'sudden infant death syndrome' (SIDS) (n = 9). The distribution patterns among the haplogroups of the disease groups (LHON, DIDMOAD and SIDS) differed considerably from the control population. LHON and DIDMOAD were significantly under-represented in the most frequent German haplogroup DC, but were concentrated in a mtDNA lineage defined by polymorphisms at nt 4216 + 11251 + 16126. As this lineage diverged into two precisely defined haplogroups, LHON and DIDMOAD could be assigned to the two haplogroups separately. Strikingly, SIDS was often found in association with two rare German haplogroups. MELAS patients were equally distributed among German haplogroups and, moreover, did not reveal any accumulation of specific D loop variants. We conclude that certain European mtDNA haplogroups define a genetic susceptibility basis for various disorders.

Mutations of SURF‐1, a gene located on chromosome 9q34, have recently been identified in patients affected by Leigh syndrome (LS), associated with deficiency of cytochrome c oxidase (COX), the terminal component of the mitochondrial respiratory chain. To investigate to what extent SURF‐1 is responsible for human disorders because of COX deficiency, we undertook sequence analysis of the SURF‐1 gene in 46 unrelated patients. We analyzed 24 COX‐defective patients classified as having typical Leigh syndrome (LSCOX), 6 patients classified as Leigh‐like (LLCOX) cases, and 16 patients classified as non‐LSCOX cases. Frameshift, stop, and splice mutations of SURF‐1 were detected in 18 of 24 (75%) of the LSCOX cases. No mutations were found in the LLCOX and non‐LSCOX group of patients. Rescue of the COX phenotype was observed in transfected cells from patients harboring SURF‐1 mutations, but not in transfected cell lines from 2 patients in whom no mutations were detected by sequence analysis. Loss of function of SURF‐1 protein is specifically associated with LSCOX, although a proportion of LSCOX cases must be the result of abnormalities in genes other than SURF‐1. SURF‐1 is the first nuclear gene to be consistently mutated in a major category of respiratory chain defects. DNA analysis can now be used to accurately diagnose LSCOX, a common subtype of Leigh syndrome. Ann Neurol 1999;46:161–166

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Tim8 and Tim13 of yeast belong to a family of evolutionary conserved zinc finger proteins that are organized in hetero-oligomeric complexes in the mitochondrial intermembrane space. Mutations in DDP1 (deafness dystonia peptide 1), the human homolog of Tim8, are associated with the Mohr-Tranebjaerg syndrome, a progressive neurodegenerative disorder. We show that DDP1 acts with human Tim13 in a complex in the intermembrane space. The DDP1⅐hTim13 complex is in direct contact with translocation intermediates of human Tim23 in mammalian mitochondria. The human DDP1⅐hTim13 complex complements the function of the TIM8⅐13 complex in yeast and facilitates import of yeast and human Tim23. Thus, the pathomechanism underlying the Mohr-Tranebjaerg syndrome may involve an impaired biogenesis of the human TIM23 complex causing severe pleiotropic mitochondrial dysfunction.The vast majority of mitochondrial proteins are encoded as precursors in the nuclear genome. Mitochondrial biogenesis is, therefore, dependent on the import and sorting of the nuclear encoded precursor proteins into mitochondrial subcompartments. In eukaryotes three distinct preprotein import systems located in the mitochondrial outer and inner membrane have been described (1-5). The outer membrane contains a general preprotein translocase, the TOM 1 complex, which mediates the recognition and binding of preproteins and their transfer across the outer membrane. This complex is most likely used by all nuclear encoded precursors. Import into and across the inner membrane is mediated by two distinct inner membrane translocases, the TIM22 and the TIM23 complexes. Both TIM complexes cooperate with the TOM complex but differ in their substrate specificity (6 -11). The TIM23 complex mediates import of preproteins with a positively charged matrix targeting signal into the mitochondrial matrix space and into the inner membrane (6, 12, 13). The translocation of such precursors into the matrix requires the membrane potential ⌬ across the inner membrane and ATP in the matrix. The ⌬ drives the translocation of the presequences through the protein-conducting channel of the TIM23 complex which is formed by the membrane-integrated proteins Tim23 and Tim17 (6,12). A molecular motor that is attached to the inner side of this channel then promotes further translocation of the mature portion of the preproteins into the matrix. This motor consists of the peripheral membrane protein Tim44, the mitochondrial Hsp70, and the nucleotide exchange factor Mge1. Together, these components in repeated ATP-dependent reaction cycles facilitate the vectorial translocation into the matrix in a stepwise manner (14).The TIM22 complex mediates the insertion of a class of hydrophobic proteins with internal targeting signals into the inner membrane (7)(8)(9)(10)(11)(15)(16)(17). Typical substrates are members of the mitochondrial carrier family and other integral inner membrane proteins that are synthesized without a matrix-targeting signal. Insertion of these precursors into the inner membrane is s...

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP produ...

The Mohr^Tranebjaerg syndrome (MTS), a neurodegenerative syndrome characterized by progressive sensorineural hearing loss, dystonia, mental retardation and blindness, is a mitochondrial disease caused by mutations in the deafness/ dystonia peptide 1 (DDP1) gene. DDP1 shows similarity to the yeast proteins Tim9, Tim10 and Tim12, components of the mitochondrial import machinery for carrier proteins. Here, we show that DDP1 belongs to a large family of evolutionarily conserved proteins. We report the identification, chromosomal localization and expressional analysis of six human family members which represent further candidate genes for neurodegenerative diseases.z 1999 Federation of European Biochemical Societies.