TM7SF1 (GPR137B): a novel lysosome integral membrane protein

In the previous proteomic study of human placenta, transmembrane 7 superfamily member 1 (TM7SF1) was found enriched in lysosome compartments. TM7SF1 encodes a 399-amino acid protein with a calculated molecular mass of 45 kDa. Bioinformatic analysis of its amino acid sequence showed that it is a multipass transmembrane protein containing a potential dileucine-based lysosomal targeting signal and four putative N-glycosylation sites. By percoll-gradient centrifugation and further subfraction ways, the lysosomal solute and membrane compartments were isolated respectively. Immunoblotting analysis indicated that TM7SF1 was co-fractioned with lysosome associated membrane protein 2 (LAMP2), which was only detected in lysosomal membrane compartments whereas not detected in the solute compartments. Using specific anti-TM7SF1 antibody and double-immunofluorescence with lysosome membrane protein LAMP1 and Lyso-Tracker Red, the colocalisations of endogenous TM7SF1 with lysosome and late endosome markers were demonstrated. All of this indicated that TM7SF1 is an integral lysosome membrane protein. Rat ortholog of TM7SF1 was found to be strongly expressed in heart, liver, kidney and brain while not or low detected in other tissues. In summary, TM7SF1 was a lysosomal integral membrane protein that shows tissue-specific expression. As a G-protein-coupled receptor in lysosome membrane, TM7SF1 was predicted function as signal transduction across lysosome membrane.     

Identification of a new Bradyrhizobium japonicum gene (frxA) encoding a ferredoxinlike protein.

An open reading frame of 74 codons was identified downstream of the nifB gene of Bradyrhizobium japonicum 110. The predicted amino acid sequence shared 63% similarity with the Rhodopseudomonas palustris ferredoxin I sequence. We propose to name the gene frxA. The frxA gene was found to be cotranscribed with the nifB gene. An insertion mutation within frxA hardly affected nitrogen fixation activity.     

Sequence of the Saccharomyces cerevisiae YTP1 gene encoding a deduced novel type-III integral membrane protein with domains of sequence similarity to mitochondrial electron-transport enzymes

The nucleotide sequence is reported for the Saccharomyces cerevisiae YTP1 (yeast putative transmembrane (TM) protein) gene, encoding a novel deduced protein of 459 amino acids (aa) in length (51643 Da). The Ytpl protein appears by computer analysis (hydropathy plots in conjunction with the combined predictions of several Internet on-line programs that deduce protein structure from primary sequence data) to be a type-III integral TM protein containing 10 or 11 TM-spanning domains. Blocks of aa sequence similarity, predominantly to mitochondrial electron transport proteins, are consistent with the notion that Ytpl is an integral TM protein and may reflect some aspect of its functional role. The C terminus of Ytpl is both hydrophilic and highly negatively charged, with 11 of the last 33 aa corresponding to Glu or Asp. Although Northern blot analysis indicates that this gene is expressed, a disruption of YTP1 shows that it is not essential. YTP1 is located between SIN4 (TSF3) and KEX2 (SRB1) at position 205 (kb) on the chromosome XIV physical map     

Identification of a novel gene (ADPRTL1) encoding a potential Poly(ADP-ribosyl)transferase protein

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein-protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.     

PAS3, a Saccharomyces cerevisiae gene encoding a peroxisomal integral membrane protein essential for peroxisome biogenesis

Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau, 1989, Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3-mutants enabled us to clone the PAS3 gene by functional complementation. DNA sequence analysis revealed a 50.6-kD protein with at least one domain of sufficient length and hydrophobicity to span a lipid bilayer. To verify these predictions antibodies were raised against a truncated portion of the PAS3 coding region overexpressed in E. coli. Pas3p was identified as a 48 kD peroxisomal integral membrane protein. It is shown that a lack of this protein causes the peroxisome-deficient phenotype and the cytosolic mislocalization of peroxisomal matrix enzymes. Based on protease digestion experiments Pas3p is discussed to be anchored in the peroxisomal membrane by its amino-terminus while the bulk of the molecule is exposed to the cytosol. These findings are consistent with the possibility that Pas3p is one component of the peroxisomal import machinery.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

The human polycystin-2 protein represents an integral membrane protein with six membrane-spanning domains and intracellular N- and C-termini

PKD2 is one of the two genes mutated in ADPKD (autosomal-dominant polycystic kidney disease). The protein product of PKD2, polycystin-2, functions as a non-selective cation channel in the endoplasmic reticulum and possibly at the plasma membrane. Hydrophobicity plots and its assignment to the TRP (transient receptor potential) family of cation channels suggest that polycystin-2 contains six transmembrane domains and that both the N- and C-termini extend into the cytoplasm. However, no experimental evidence for this model has so far been provided. To determine the orientation of the different loops of polycystin-2, we truncated polycystin-2 within the predicted loops 1-5 and tagged the constructs at the C-terminus with an HA (haemagglutinin) epitope. After transient expression and selective membrane permeabilization, immunofluorescence staining for the HA epitope revealed that loops 1, 3 and 5 extend into the lumen of the endoplasmic reticulum or the extracellular space, whereas loops 2 and 4 extend into the cytoplasm. This approach also confirmed the cytoplasmic orientation of the N- and C-termini of polycystin-2. In accordance with the immunofluorescence data, protease protection assays from microsomal preparations yielded protected fragments when polycystin-2 was truncated in loops 1, 3 and 5, whereas no protected fragments could be detected when polycystin-2 was truncated in loops 2 and 4. The results of the present study therefore provide the first experimental evidence for the topological orientation of polycystin-2.     

Molecular cloning and sequence analysis of the gene encoding OmpL1, a transmembrane outer membrane protein of pathogenic Leptospira spp.

Pathogenic Leptospira spp. are spirochetes that have a low transmembrane outer membrane protein content relative to that of enteric gram-negative bacteria. In a previous study we identified a 31-kDa surface protein that was present in strains of Leptospira alstoni in amounts which correlated with the outer membrane particle density observed by freeze fracture electron microscopy (D. A. Haake, E. M. Walker, D. R. Blanco, C. A. Bolin, J. N. Miller, and M. A. Lovett, Infect. Immun. 59:1131-1140, 1991). The N-terminal amino acid sequence was used to design a pair of oligonucleotides which were utilized to screen a lambda ZAP II library containing EcoRI fragments of L. alstoni DNA. A 2.5-kb DNA fragment which contained the entire structural ompL1 gene was identified. The structural gene deduced from the sequence of this DNA fragment would encode a 320-amino-acid polypeptide with a 24-amino-acid leader peptide and a leader peptidase I cleavage site. Processing of OmpL1 results in a mature protein with a predicted molecular mass of 31,113 Da. Secondary-structure prediction identified repeated stretches of amphipathic beta-sheets typical of outer membrane protein membrane-spanning sequences. A topological model of OmpL1 containing 10 transmembrane segments is suggested. A recombinant OmpL1 fusion protein was expressed in Escherichia coli in order to immunize rabbits with the purified protein. Upon Triton X-114 extraction of L. alstoni and phase separation, anti-OmpL1 antiserum recognized a single band on immunoblots of the hydrophobic detergent fraction which was not present in the hydrophilic aqueous fraction. Immunoelectron microscopy with anti-OmpL1 antiserum demonstrates binding to the surface of intact L. alstoni. DNA hybridization studies indicate that the ompL1 gene is present in a single copy in all pathogenic Leptospira species that have been tested and is absent in nonpathogenic Leptospira species. OmpL1 may be the first spirochetal transmembrane outer membrane protein for which the structural gene has been cloned and sequenced.     

phoR1, a gene encoding a new histidine protein kinase Myxococcus xanthus

A soil bacterium able to undergo multicellular development and a coordinated gliding in swarms, requires an accurate regulatory network of phosphorelay proteins. Inorganic phosphate is a limiting nutrient in soil and its importance in regulation is critical. As a step towards studying phosphate regulation and its influence in the developmental process in this bacterium, we screened a Myxococcus xanthus library for clones with phosphatase activity, and found four different ones. The deduced sequence of one of the cloned inserts is similar to that of the classic transmembrane histidine protein kinase of the sensor family of the two-component signal transduction systems with a high sequence similarity to the sensor kinase in the Pho regulon of Bacillus subtilis PhoR. This gene has been named phoR1 and its deduced amino acid sequence consists of 455 residues with a predicted molecular mass of 48.5 kDa. The M. xanthus PhoR1 deduced sequence contains all the characteristic histidine protein kinase motifs in the same order and with the same spacing. A hydropathy profile indicates two membrane-spanning segments located at the extreme N-terminus, according to the putative sensor role of this domain. A gene-disrupted mutant is unable to produce normal mature fruiting bodies and produces fewer spores. This revised version was published online in June 2006 with corrections to the Cover Date.     

The novel gene encoding a putative transmembrane protein is mutated in gnathodiaphyseal dysplasia (GDD)

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.     

Functional analysis of the transmembrane (TM) domain of the Autographa californica multicapsid nucleopolyhedrovirus GP64 protein: substitution of heterologous TM domains

GP64, the major envelope glycoprotein of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) budded virion, is important for host cell receptor binding and mediates low-pH-triggered membrane fusion during entry by endocytosis. In the current study, we examined the functional role of the AcMNPV GP64 transmembrane (TM) domain by replacing the 23-amino-acid GP64 TM domain with corresponding TM domain sequences from a range of viral and cellular type I membrane proteins, including Orgyia pseudotsugata MNPV (OpMNPV) GP64 and F, thogotovirus GP75, Lymantria dispar MNPV (LdMNPV) F, human immunodeficiency virus type 1 (HIV-1) GP41, human CD4 and glycophorin A (GpA), and influenza virus hemagglutinin (HA), and with a glycosylphosphatidylinositol (GPI) anchor addition sequence. In transient expression experiments with Sf9 cells, chimeric GP64 proteins containing either a GPI anchor or TM domains from LdMNPV F or HIV-1 GP41 failed to localize to the cell surface and thus appear to be incompatible with either GP64 structure or cell transport. All of the mutant constructs detected at the cell surface mediated hemifusion (outer leaflet merger) upon low-pH treatment, but only those containing TM domains from CD4, GpA, OpMNPV GP64, and thogotovirus GP75 mediated pore formation and complete membrane fusion activity. This supports a model in which partial fusion (hemifusion) proceeds by a mechanism that is independent of the TM domain and the TM domain participates in the enlargement or expansion of fusion pores after hemifusion. GP64 proteins containing heterologous TM domains mediated virion budding with dramatically differing levels of efficiency. In addition, chimeric GP64 proteins containing TM domains from CD4, GpA, HA, and OpMNPV F were incorporated into budded virions but were unable to rescue the infectivity of a gp64 null virus, whereas those with TM domains from OpMNPV GP64 and thogotovirus GP75 rescued infectivity. These results show that in addition to its basic role in membrane anchoring, the GP64 TM domain is critically important for GP64 trafficking, membrane fusion, virion budding, and virus infectivity. These critical functions were replaced only by TM domains from related viral membrane proteins.     

LMP1 transmembrane domain 1 and 2 (TM1-2) FWLY mediates intermolecular interactions with TM3-6 to activate NF-kappaB

The Epstein-Barr virus oncoprotein LMP1 has six transmembrane domains (TMs) that enable intermolecular aggregation and constitutive signaling through two C-terminal cytosolic domains. Expression of both TMs 1 and 2 without the C terminus (TM1-2DeltaC) and TMs 3 to 6 fused to the C terminus (TM3-6) results in partial association, which is substantially decreased by TM1 F38WLY41 mutation to A38ALA41. We now investigate whether TM1-2DeltaC can functionally interact with TM3-6. TM1-2DeltaC induced TM3-6 to mediate NF-kappaB activation at 59% of LMP1 levels, and the effect was dependent on TM1-2 F38WLY41. TM1-2DeltaC even induced TM3-4 C terminus-mediated NF-kappaB activation to 44% of LMP1 levels. Surprisingly, this effect was TM1 F38WLY41 independent, indicative of a role for TMs 5 and 6 in TM1 F38WLY41 effects. TM3 W98 was also important for TM1-2DeltaC induction of TM3-6-mediated NF-kappaB activation, for association, and for TM1 F38WLY41 dependence on C-terminal NF-kappaB activation. These data support models in which the TM1 F38WLY41 effects are at least partially dependent on TM3 W98 and a residue(s) in TMs 5 and 6.     

SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein

In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5 kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130 kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95 kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression.