The 16 kDa subunit of vacuolar H+-ATPase is a novel sarcoglycan-interacting protein

The sarcoglycan complex in muscle consists of alpha-, beta-, gamma- and delta-sarcoglycan and is part of the larger dystrophin-glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between delta-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H(+)-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with delta-sarcoglycan and the highly related gamma-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of beta(1)-integrin is increased. Recent studies have shown that 16K also interacts directly with beta(1)-integrin and our data demonstrated that sarcoglycans, 16K and beta(1)-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.     

Chromosomal localization of human Na+, K+-ATPase alpha- and beta-subunit genes

Na+, K+-ATPase is a heterodimeric enzyme responsible for the active maintenance of sodium and potassium gradients across the plasma membrane. Recently, cDNAs for several tissue-specific isoforms of the larger catalytic alpha-subunit and the smaller beta-subunit have been cloned. We have hybridized rat brain and human kidney cDNA probes, as well as human genomic isoform-specific DNA fragments, to Southern filters containing panels of rodent X human somatic cell hybrid lines. The results obtained have allowed us to assign the loci for the ubiquitously expressed alpha-chain (ATP1A1) to human chromosome 1, region 1p21----cen, and for the alpha 2 isoform that predominates in neural and muscle tissues (ATP1A2) to chromosome 1, region cen----q32. A common PstI RFLP was detected with the ATP1A2 probe. The alpha 3 gene, which is expressed primarily in neural tissues (ATP1A3), was assigned to human chromosome 19. A fourth alpha gene of unknown function (alpha D) that was isolated by molecular cloning (ATP1AL1) was mapped to chromosome 13. Although evidence to date had suggested a single gene for the beta-subunit, we found hybridizing restriction fragments derived from two different human chromosomes. On the basis of knowledge of conserved linkage groups on human and murine chromosomes, we propose that the coding gene ATP 1B is located on the long arm of human chromosome 1 and that the sequence on human chromosome 4 (ATP 1BL1) is either a related gene or a pseudogene.     

Chromosomal localization of the mouse gene coding for the 68 kDa neurofilament subunit

The chromosomal localization of the mouse gene coding for the 68 kDa intermediate filament subunit of neurones (NF-L) was determined by in situ hybridization using specific 3H-labelled DNA probes. There is only one copy of the NF-L gene. The gene encoding NF-L is located on chromosome 14 region (D1-E1).     

The 16 kDa subunit of vacuolar H+-ATPase is a novel sarcoglycan-interacting protein

The sarcoglycan complex in muscle consists of α-, β-, γ- and δ-sarcoglycan and is part of the larger dystrophin–glycoprotein complex (DGC), which is essential for maintaining muscle membrane integrity. Mutations in any of the four sarcoglycans cause limb-girdle muscular dystrophies (LGMD). In this report, we have identified a novel interaction between δ-sarcoglycan and the 16 kDa subunit c (16K) of vacuolar H⁺-ATPase. Co-expression studies in heterologous cell system revealed that 16K interacts specifically with δ-sarcoglycan and the highly related γ-sarcoglycan through the transmembrane domains. In cultured C2C12 myotubes, 16K forms a complex with sarcoglycans at the plasma membrane. Loss of sarcoglycans in the sarcoglycan-deficient BIO14.6 hamster destabilizes the DGC and alters the localization of 16K at the sarcolemma. In addition, the steady state level of β₁-integrin is increased. Recent studies have shown that 16K also interacts directly with β₁-integrin and our data demonstrated that sarcoglycans, 16K and β₁-integrin were immunoprecipitated together in C2C12 myotubes. Since sarcoglycans have been proposed to participate in bi-directional signaling with integrins, our findings suggest that 16K might mediate the communication between sarcoglycans and integrins and play an important role in the pathogenesis of muscular dystrophy.     

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromosomal localization of 13 candidate genes for human obesity in the pig genome

Thirteen candidate genes for human obesity were selected for cytogenetic mapping by FISH in the pig genome. Among them, 6 genes were assigned to chromosomes for the first time (NR3C1, GNB3, ADRB1, ADRB2, ADRB3 andUCP1). Location of the other 7 genes (INSIG2, LIPIN1, PLIN, NAMPT, ADIPOQ, UCP2 andUCP3), earlier mapped by somatic cell hybridization or with the use of a radiation hybrid panel, was verified (INSIG2) or more precisely described. The genes were assigned to the following chromosomes:INSIG2 to SSC15q12,LIPIN1 to SSC3q26,NR3C1 to SSC2q29,PLIN to SSC7q15,GNB3 to SSC5q21,NAMPT to SSC9q23,ADIPOQ to SSC13q41,ADRB1 to SSC14q28,ADRB2 to SSC2q29,ADRB3 to SSC15q13-14,UCP1 to SSC8q21-22, and bothUCP2 andUCP3 to SSC9p24. Most of the genes were located within known QTL for pig fatness traits.     

Chromosomal localization of three pulmonary surfactant protein genes in the mouse.

Pulmonary surfactant, a protein-phospholipid mixture, maintains surface tension at the lung epithelium/air interface preventing alveolar collapse during respiration. For mammals appropriate developmental production of surfactant is necessary for adaptation to the air breathing environment. Deficiency of pulmonary surfactant results in respiratory distress syndrome (RDS), a leading cause of death in premature infants. Recently, three lung-specific pulmonary surfactant proteins designated SP-A, SP-B, and SP-C have been described. Cloned sequences for the genes that encode each of these proteins have been partially characterized in humans and other species. Analysis of interspecific backcross mice has allowed us to map the chromosomal locations of these three genes in the mouse. The gene encoding SP-A (Sftp-1) and the gene encoding SP-C (Sftp-2) both map to mouse chromosome 14, although at separate locations, while the gene encoding SP-B (Sftp-3) maps to chromosome 6. The mouse map locations determined in this study for the Sftp genes are consistent with the locations of these genes on the human genetic map and the syntenic relationships between the human and the mouse genomes.     

Vacuolar type H(+)-ATPase genes: presence of four genes including pseudogenes for the 16-kDa proteolipid subunit in the human genome.

Genes for the human vacuolar type H(+)-ATPase proteolipid (16-kDa) subunit were cloned and their nucleotide sequences were determined. Comparison of the deduced sequences indicated that at least four genes including pseudogenes are present in the human genome. One of them corresponded to that for the 16-kDa subunit expressed in HeLa cells. The coding sequence was separated by two introns. The second intron was located in the DNA segment giving a loop between the second and third transmembrane helices, supporting the idea that the 16-kDa subunit was evolved by gene duplication. The primary sequence determined from the second clone had a termination codon behind the third transmembrane helix. Possible translation products from the other two clones had no putative acidic residues essential for proton transport function of the 16-kDa subunit. Thus, it is interesting to know whether these genes are transcribed, since they may have unique cellular functions.     

Chromosomal localization of three human poly(A)-binding protein genes and four related pseudogenes

In humans, the poly(A)-binding proteins (PABPs) comprise a small nuclear isoform and a conserved gene family that displays at least three functional proteins: PABP1, inducible PABP (iPABP), and PABP3, plus four pseudogenes (1, 2, 3, and PABP4). In situ hybridization of PABP3 cDNA as the probe on metaphasic chromosomes have revealed five possible loci for this gene family at 2q21-q22, 13q11-q12, 12q13.3-q15, 8q22, and 3q24-q25. Amplifications of specific DNA fragments from a human-rodent somatic cell hybrid panel have allowed us to associate PABP1 and PABP3 with 8q22 and 13q11-q12, respectively. The iPABP gene has been assigned to chromosome 1. This result, compared with radiation hybrid database information, strengthens the location of this gene to 1p32-p36. The pseudogenes PABP4, 1, and 2 have been assigned to chromosomes 15, 4, and 14, respectively. Three loci detected on chromosome spreads are not associated with any amplified fragment. They might represent other related PABP genes not yet identified.     

Stator structure and subunit composition of the V(1)/V(0) Na(+)-ATPase of the thermophilic bacterium Caloramator fervidus

The V-type Na(+)-ATPase of the thermophilic, anaerobic bacterium Caloramator fervidus was purified to homogeneity. The subunit compositions of the catalytic V(1) and membrane-embedded V(0) parts were determined and the structure of the enzyme complex was studied by electron microscopy. The V(1) headpiece consists of seven subunits present in one to three copies, and the V(0) part of two subunits in a ratio of 5:2. An analysis of over 7500 single particle images obtained by electron microscopy of the purified V(1)V(0) enzyme complex revealed that the stalk region, connecting the V(1) and V(0) parts, contains two peripheral stalks in addition to a central stalk. One of the two is connected to the V(0) part, while the other is connected to the first via a bar-like structure that is positioned just above V(0), parallel with the plane of the membrane. In projection, this bar seems to contact the central stalk. The data show that the stator structure that prevents rotation of the static part of V(0) relative to V(1) in the rotary catalysis mechanism of energy coupling in ATPases/ATPsynthases is more complex than previously thought.     

Chromosomal localization of seven neuronal nicotinic acetylcholine receptor subunit genes in humans.

We have determined the chromosomal location of seven human neuronal nicotinic acetylcholine receptor subunit genes by genomic Southern analysis of hamster/human somatic cell hybrid DNAs. The beta 2 subunit gene was localized to human chromosome 1, the alpha 2 and beta 3 subunit genes were localized to human chromosome 8, the alpha 3, alpha 5, and beta 4 subunit genes were localized to human chromosome 15, and the alpha 4 subunit gene was localized to human chromosome 20. Mapping of the beta 2 subunit gene to chromosome 1 establishes a syntenic group with the amylase gene locus on human chromosome 1 and mouse chromosome 3, while mapping of the alpha 3 subunit gene to chromosome 15 confirms the existence of a syntenic group with the mannose phosphate isomerase gene locus on human chromosome 15 and mouse chromosome 9.     

Differential localization of vacuolar H+-ATPases containing a1, a2, a3, or a4 (ATP6V0A1-4) subunit isoforms along the nephron.

Vacuolar H(+)-ATPase are multi-subunit containing pumps important for several processes along the nephron such as receptor mediated endocytosis, acidification of intracellular organelles, bicarbonate reabsorption and secretion, and H(+)- extrusion. Mutations in the human a4 (ATP6V0A4) subunit cause distal renal tubular acidosis (dRTA). There are 4 known isoforms of the 'a' subunit (a1-a4). Here we investigated the expression and localization of all four isoforms in mouse kidney. Real-time PCR detected mRNAs encoding all four 'a' isoforms in mouse kidney with a relative abundance in the following order: a4>a2=a1>a3. Immunolocalization demonstrated expression of all 'a' subunits in the proximal tubule and in the intercalated cells of the collecting system. In intercalated cells a1 and a4 isoforms appeared on both the apical and basolateral side and were expressed in all subtypes of intercalated cells. In contrast, a2, and a3 were only found in the apical membrane. a1 and a4 were colocalized in the same cells with AE1 or pendrin, whereas a2 was only found in AE1 positive cells but absent from pendrin expressing intercalated cells. These results suggest that vacuolar H(+)-ATPases containing different 'a' isoforms may serve specific and distinct functions and may help explaining why loss of the a4 isoform causes only dRTA without an apparent defect in the proximal tubule.     

Chromosomal localization of the gene coding for alpha-subunit of Na+,K+-ATPase in the American mink (Mustela vison)

The gene coding for the alpha-subunit of Na+,K+-ATPase has been localized on chromosome 2 of the American mink (Mustela vison) using the somatic cell hybrids mink-Chinese hamster and pig cDNA clones as hybridization probes.     

Chromosomal localization of the gene coding for the beta-subunit of Na+,K+-ATPase in the American mink (Mustela vison)

The BATP gene coding for the beta-subunit of Na+,K+-ATPase has been localized on chromosome 13 of the American mink (Mustela vison) using mink-Chinese hamster somatic cell hybrids and pig cDNA clones as probes. The AATP gene for the alpha-subunit of Na+,K+-ATPase is on mink chromosome 2 [(1987) FEBS Lett. 217, 42-44]. Consequently, the AATP and BATP genes for the Na+,K+-ATPase occupy separate mink chromosomes.