The LETM1/YOL027 gene family encodes a factor of the mitochondrial K+ homeostasis with a potential role in the Wolf-Hirschhorn syndrome

The yeast open reading frames YOL027 and YPR125 and their orthologs in various eukaryotes encode proteins with a single predicted trans-membrane domain ranging in molecular mass from 45 to 85 kDa. Hemizygous deletion of their human homolog LETM1 is likely to contribute to the Wolf-Hirschhorn syndrome phenotype. We show here that in yeast and human cells, these genes encode integral proteins of the inner mitochondrial membrane. Deletion of the yeast YOL027 gene (yol027Delta mutation) results in mitochondrial dysfunction. This mutant phenotype is complemented by the expression of the human LETM1 gene in yeast, indicating a functional conservation of LetM1/Yol027 proteins from yeast to man. Mutant yol027Delta mitochondria have increased cation contents, particularly K+ and low-membrane-potential Deltapsi. They are massively swollen in situ and refractory to potassium acetate-induced swelling in vitro, which is indicative of a defect in K+/H+ exchange activity. Thus, YOL027/LETM1 are the first genes shown to encode factors involved in both K+ homeostasis and organelle volume control.     

LETM1, a novel gene encoding a putative EF-hand Ca(2+)-binding protein, flanks the Wolf-Hirschhorn syndrome (WHS) critical region and is deleted in most WHS patients.

Deletions within human chromosome 4p16.3 cause Wolf-Hirschhorn syndrome (WHS), which is characterized by severe mental and developmental defects. It is thought that haploinsufficiency of more than one gene contributes to the complex phenotype. We have cloned and characterized a novel gene (LETM1) that is deleted in nearly all WHS patients. LETM1 encodes a putative member of the EF-hand family of Ca(2+)-binding proteins. The protein contains two EF-hands, a transmembrane domain, a leucine zipper, and several coiled-coil domains. On the basis of its possible Ca(2+)-binding property and involvement in Ca(2+) signaling and/or homeostasis, we propose that haploinsufficiency of LETM1 may contribute to the neuromuscular features of WHS patients.     

Sex-specific expression of an evolutionarily conserved male regulatory gene, DMRT1, in birds

Based on its Z-sex-chromosomal location and its structural homology to male sexual regulatory factors in humans (DMRT1 and DMRT2), Drosophila (Dsx), and Caenorhabditis elegans (Mab-3), chicken DMRT1 is an excellent candidate for a testis-determining factor in birds. The data we present provide further strong support for this hypothesis. By whole mount in situ hybridization chicken DMRT1 is expressed at higher levels in the male than in the female genital ridges during early stages of embryogenesis. Its expression becomes testis-specific after onset of sexual differentiation. Northern blot and RT PCR analysis showed that in adult birds DMRT1 is expressed exclusively in the testis. We propose that two gene dosages are required for testis formation in ZZ males, whereas expression from a single Z chromosome in ZW females leads to female sexual differentiation.     

LETM1 haploinsufficiency causes mitochondrial defects in cells from humans with Wolf-Hirschhorn syndrome: implications for dissecting the underlying pathomechanisms in this condition

Wolf-Hirschhorn syndrome (WHS) represents an archetypical example of a contiguous gene deletion disorder – a condition comprising a complex set of developmental phenotypes with a multigenic origin. Epileptic seizures, intellectual disability, growth restriction, motor delay and hypotonia are major co-morbidities in WHS. Haploinsufficiency of LETM1, which encodes a mitochondrial inner-membrane protein functioning in ion transport, has been proposed as an underlying pathomechanism, principally for seizures but also for other core features of WHS, including growth and motor delay. Growing evidence derived from several model organisms suggests that reduced LETM1 expression is associated with some element of mitochondrial dysfunction. Surprisingly, LETM1-dependent mitochondrial functional deficits have not previously been described in cells from individuals with WHS. Here, using a unique panel of WHS-patient-derived cell lines with deletions of differing sizes, incorporating LETM1 or not, we show, for the first time, that LETM1 expression is reduced in mitochondria isolated from WHS-patient cells. Furthermore, we show that this is associated with distinct mitochondrial phenotypes, including altered intracellular [Ca²⁺] levels, dysfunctional mitochondrial transition-pore opening, hyperpolarization and superoxide leakage from resting mitochondria. Interestingly, we find that these phenotypes segregate with seizures in our WHS cohort. Our findings identify novel cellular phenotypes in WHS attributable to a 50% reduction in LETM1 expression level; these phenotypes could underlie and/or contribute to some of the core clinical features of this condition.KEY WORDS: LETM1, Wolf-Hirschhorn syndrome, Mitochondria     

Transmembrane topology of pmt1p, a member of an evolutionarily conserved family of protein O-mannosyltransferases

The identification of the evolutionarily conserved family of dolichyl-phosphate-D-mannose:protein O-mannosyltransferases (Pmts) revealed that protein O-mannosylation plays an essential role in a number of physiologically important processes. Strikingly, all members of the Pmt protein family share almost identical hydropathy profiles; a central hydrophilic domain is flanked by amino- and carboxyl-terminal sequences containing several putative transmembrane helices. This pattern is of particular interest because it diverges from structural models of all glycosyltransferases characterized so far. Here, we examine the transmembrane topology of Pmt1p, an integral membrane protein of the endoplasmic reticulum, from Saccharomyces cerevisiae. Structural predictions were directly tested by site-directed mutagenesis of endogenous N-glycosylation sites, by fusing a topology-sensitive monitor protein domain to carboxyl-terminal truncated versions of the Pmt1 protein and, in addition, by N-glycosylation scanning. Based on our results we propose a seven-transmembrane helical model for the yeast Pmt1p mannosyltransferase. The Pmt1p amino terminus faces the cytoplasm, whereas the carboxyl terminus faces the lumen of the endoplasmic reticulum. A large hydrophilic segment that is oriented toward the lumen of the endoplasmic reticulum is flanked by five amino-terminal and two carboxyl-terminal membrane spanning domains. We could demonstrate that this central loop is essential for the function of Pmt1p.     

Extended reading frame of a ubiquitin gene encodes a stable, conserved, basic protein

Antibodies specific for the 80-amino acid hypothetical protein encoded by the in-frame, 3'-extension of a human ubiquitin gene were produced in rabbits by immunization with a 14-residue synthetic peptide. When used to probe HeLa cell extracts for the non-ubiquitin product of this natural fusion gene, the antipeptide sera detected a protein with an apparent molecular weight of 16,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. An immunoreactive protein of identical mobility was detected in organisms ranging from Acanthamoeba to man, indicating that the extension protein, like ubiquitin, is highly conserved. The immunoreactive protein was isolated from calf thymus, and direct sequencing revealed the first 16 amino acids to be identical to those predicted from the extension portion of the human cDNA. Thus, ubiquitin was no longer present at the amino terminus. The purified bovine extension protein failed to react with a ubiquitin-specific antibody indicating the absence of isopeptide-linked ubiquitin as well. Moreover, by denaturing gel permeation chromatography the extension has a molecular weight of 10,000 Da, a value that corresponds more closely to the size of the extension alone (9,000 Da) than to the intact fusion protein (17,500 Da). The extension protein, which was found in both cytoplasmic and nuclear fractions of HeLa cells, persisted at high levels when protein synthesis was blocked with cycloheximide or puromycin. These results show that the 80-residue extension protein is the stable, processed product of the ubiquitin fusion gene.     

Saccharomyces cerevisiae SMT4 encodes an evolutionarily conserved protease with a role in chromosome condensation regulation

In a search for regulatory genes affecting the targeting of the condensin complex to chromatin in Saccharomyces cerevisiae, we identified a member of the adenovirus protease family, SMT4. SMT4 overexpression suppresses the temperature-sensitive conditional lethal phenotype of smc2-6, but not smc2-8 or smc4-1. A disruption allele of SMT4 has a prominent chromosome phenotype: impaired targeting of Smc4p-GFP to rDNA chromatin. Site-specific mutagenesis of the predicted protease active site cysteine and histidine residues of Smt4p abolishes the SMT4 function in vivo. The previously uncharacterized SIZ1 (SAP and Miz) gene, which encodes a protein containing a predicted DNA-binding SAP module and a Miz finger, is identified as a bypass suppressor of the growth defect associated with the SMT4 disruption. The SIZ1 gene disruption is synthetically lethal with the SIZ2 deletion. We propose that SMT4, SIZ1, and SIZ2 are involved in a novel pathway of chromosome maintenance.     

An abundant evolutionarily conserved CSB-PiggyBac fusion protein expressed in Cockayne syndrome

Cockayne syndrome (CS) is a devastating progeria most often caused by mutations in the CSB gene encoding a SWI/SNF family chromatin remodeling protein. Although all CSB mutations that cause CS are recessive, the complete absence of CSB protein does not cause CS. In addition, most CSB mutations are located beyond exon 5 and are thought to generate only C-terminally truncated protein fragments. We now show that a domesticated PiggyBac-like transposon PGBD3, residing within intron 5 of the CSB gene, functions as an alternative 3′ terminal exon. The alternatively spliced mRNA encodes a novel chimeric protein in which CSB exons 1–5 are joined in frame to the PiggyBac transposase. The resulting CSB-transposase fusion protein is as abundant as CSB protein itself in a variety of human cell lines, and continues to be expressed by primary CS cells in which functional CSB is lost due to mutations beyond exon 5. The CSB-transposase fusion protein has been highly conserved for at least 43 Myr since the divergence of humans and marmoset, and appears to be subject to selective pressure. The human genome contains over 600 nonautonomous PGBD3-related MER85 elements that were dispersed when the PGBD3 transposase was last active at least 37 Mya. Many of these MER85 elements are associated with genes which are involved in neuronal development, and are known to be regulated by CSB. We speculate that the CSB-transposase fusion protein has been conserved for host antitransposon defense, or to modulate gene regulation by MER85 elements, but may cause CS in the absence of functional CSB protein.     

A highly evolutionarily conserved mitochondrial protein is structurally related to the protein encoded by the Escherichia coli groEL gene.

We recently reported that a Tetrahymena thermophila 58-kilodalton (kDa) mitochondrial protein (hsp58) was selectively synthesized during heat shock. In this study, we show that hsp58 displayed antigenic similarity with mitochondrially associated proteins from Saccharomyces cerevisiae (64 kDa), Xenopus laevis (60 kDa), Zea mays (62 kDa), and human cells (59 kDa). Furthermore, a 58-kDa protein from Escherichia coli also exhibited antigenic cross-reactivity to an antiserum directed against the T. thermophila mitochondrial protein. The proteins from S. cerevisiae and E. coli antigenically related to hsp58 were studied in detail and found to share several other characteristics with hsp58, including heat inducibility and the property of associating into distinct oligomeric complexes. The T. thermophila, S. cerevisiae, and E. coli macromolecular complexes containing these related proteins had similar sedimentation characteristics and virtually identical morphologies as seen with the electron microscope. The distinctive properties of the E. coli homolog to T. thermophila hsp58 indicate that it is most likely the product of the groEL gene.     

RAP55: insights into an evolutionarily conserved protein family

The RAP55 protein family is evolutionarily conserved in eukaryotes. Two highly conserved paralogues, RAP55A and RAP55B, exist in vertebrates; their functional properties and expression patterns remain to be compared. RAP55 proteins share multiple domains: the LSm14 domain, a serine/threonine rich region, an FDF (phenylalanine-aspartate-phenylalanine) motif, an FFD-TFG box and RGG (arginine-glycine-glycine) repeats. Together these domains are responsible for RAP55 proteins participating in translational repression, incorporation into mRNP particles, protein-protein interactions, P-body formation and stress granule localisation. All RAP55A proteins localise to P-body-like complexes either in the germline or in somatic cells. Xenopus laevis RAP55B has been shown to be part of translationally repressed mRNP complexes in early oocytes. Together these findings suggest that this protein family has evolved a common and fundamental role in the control of mRNA translation. Furthermore human RAP55A is an autoantigen detected in the serum of patients with primary biliary cirrhosis (PBC). The link between RAP55A, P-bodies and PBC remains to be elucidated.     

YidC,a newly defined evolutionarily conserved protein,mediates membrane protein assembly in bacteria

Membranes contain proteins that catalyze a variety of reactions, which lead to the selective permeability of the membrane. For membrane proteins to function as receptors, transporters, channels, and ATPases, they must be targeted to their correct membrane and inserted into the lipid bilayer. Recently, a new membrane component called YidC was discovered that mediates the insertion of proteins into membranes in bacteria. YidC homologs also exist in mitochondria and chloroplasts. Depletion of YidC from the cell interferes with the insertion of membrane proteins that insert both dependent and independent of the SecYEG/SecDFYajC machinery. YidC directly interacts with membrane proteins during the membrane protein insertion process and assists in the folding of the hydrophobic regions into the membrane bilayer. The chloroplast and bacterial YidC homologs are truly functional homologs because the chloroplast homolog Alb3 functionally complements the bacterial YidC depletion strain. The role of YidC in the membrane insertion pathway will be reviewed.     

MHO1, an evolutionarily conserved gene, is synthetic lethal with PLC1; Mho1p has a role in invasive growth

The novel protein Memo (Mediator of ErbB2 driven cell motility) was identified in a screen for ErbB2 interacting proteins and found to have an essential function in cell motility. Memo is evolutionarily conserved with homologs found in all branches of life; the human and yeast proteins have a similarity of >50%. In the present study we used the model organism S. cerevisiae to characterize the Memo-homologue Mho1 (Yjr008wp) and to investigate its function in yeast. In a synthetic lethal screen we found MHO1 as a novel synthetic lethal partner of PLC1, which encodes the single phospholipase C in yeast. Double-deleted cells lacking MHO1 and PLC1, proliferate for up to ten generations. Introduction of human Memo into the memoΔplc1Δ strain rescued the synthetic lethal phenotype suggesting that yeast and human proteins have similar functions. Mho1 is present in the cytoplasm and the nucleus of yeast cells; the same distribution of Memo was found in mammalian cells. None of the Memo homologues have a characteristic nuclear localization sequence, however, a conserved nuclear export sequence is found in all. In mammalian cells, blocking nuclear export with Leptomycin B led to nuclear Memo accumulation, suggesting that it is actively exported from the nucleus. In yeast MHO1 expression is induced by stress conditions. Since invasive growth in S. cerevisiea is also stress-induced, we tested Mho1's role in this response. MHO1 deletion had no effect on invasion induced by nutrient deprivation, however, Mho1 overexpression blocked the invasive ability of yeast cells, suggesting that Mho1 might be acting in a dominant negative manner. Taken together, our results show that MHO1 is a novel synthetic lethal interactor with PLC1, and that both gene products are required for proliferation. Moreover, a role for Memo in cell motility/invasion appears to be conserved across species.