Cloning of cDNA encoding a 32-kDa protein. An accessory polypeptide of the H+-ATPase from chromaffin granules

The purified H+-ATPase from chromaffin granules is composed of several polypeptides, one of which has an apparent molecular weight of 39,000. Immunoblots with the antibody against this protein and various membrane preparations showed that similar or even identical polypeptides may be associated with the H+-ATPases from synaptic vesicle, kidney microsomes, and lysosomes. A cDNA library was constructed from bovine adrenal medulla, and the cDNA encoding the polypeptide was isolated and sequenced. Search in DNA and protein data banks revealed no significant homology to known genes. Hydrophobicity plot revealed no obvious transmembrane segments with the exception of one stretch of hydrophobic and neutral amino acid starting at leucine 16. The cDNA was shown to encode the entire polypeptide by the virtue of an amino acid sequence corresponding to the N terminus of the open reading frame and by subunit and site-specific antibodies. The cDNA was cloned into an expression vector, transcribed by T7 polymerase, and translated by reticulocyte lysate. Even though the cDNA encodes a protein with a molecular weight of 31,495, the translation product comigrated on sodium dodecyl sulfate gels with the subunit of the purified H+-ATPase. In line with several other subunits of vacuolar H+-ATPases, no signal sequence was detected in the translated gene. Northern blots revealed the presence of a single mRNA of about 1.6 kb in bovine adrenal medulla. However, liver, lung, and kidney may contain additional mRNA of about 1.7 kb.     

Cloning and mutational analysis of the gene encoding subunit C of yeast vacuolar H(+)-ATPase.

A DNA fragment containing the gene encoding subunit C of vaculor H(+)-ATPase (V-ATPase) was cloned from a yeast library. The predicted amino acid sequence indicated that the C subunit consists of 373 amino acids with a calculated molecular mass of 42,287 Da. The protein from yeast is 37% identical in its amino acid sequence to the C subunit of bovine V-ATPase. The DNA fragment that was cloned in this study contained two additional reading frames. At the 5' end an amino acid sequence that is homologous to Artemia elongation factor 1 was detected. At the 3' end the N-terminal part of a kinesin-like protein was observed. The gene encoding subunit C of the V-ATPase was interrupted, and the resulting mutant could not grow at high pH and was sensitive to low and high Ca2+ concentrations in the growth medium. Transformation of the mutant by a plasmid containing the gene encoding subunit C repaired the phenotype of the mutant. Substitution of more than half of the coding region by a corresponding DNA fragment encoding the bovine subunit C resulted in a phenotype indistinguishable from wild type. Immunological studies with the disruptant mutant revealed that subunit C is necessary for the assembly of the catalytic sector of the enzyme.     

Primary structure of V-ATPase subunit B from Manduca sexta midgut.

The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.     

Structural conservation and functional diversity of V-ATPases

The vacuolar system of eukaryotic cells contains a large number of organelles that are primary energized by an H⁺-ATPase that was named V-ATPase. The structure and function of V-ATPases from various sources was extensively studied in the last few years. Several genes encoding subunits of the enzyme were cloned and sequenced. The sequence information revealed the relations between V-ATPases and F-ATPases that evolved from common ancestral genes. The two families of proton pumps share structural and functional similarity. They contain distinct peripheral catalytic sectors and hydrophobic membrane sectors. Genes encoding subunits of V-ATPase in yeast cells were interrupted to yield mutants that are devoid of the enzyme and are sensitive to pH and calcium concentrations in the medium. The mutants were used to study structure, function, molecular biology, and biogenesis of the V-ATPase. They also shed light on the functional assembly of the enzyme in the vacuolar system.     

Sequence analysis of the catalytic subunit of H(+)-ATPase from porcine renal brush-border membranes.

The catalytic subunit of the H(+)-ATPase from brush-border membranes of porcine renal proximal tubules was labeled with the hydrophobic SH-group reagent 10-N-(bromoacetyl)amino-1-decyl-beta-glucopyranoside (BADG) which irreversibly inhibits proton pump activity in the absence but not in the presence of ATP. The labeled protein was purified and digested with proteinases. After isolation and sequencing of proteolytic peptides two BADG-labeled cysteines were identified. The amino acid sequences of the obtained proteolytic peptides were homologous to the catalytic subunit of V-ATPases. From mRNA of porcine kidney cortex a catalytic H(+)-ATPase subunit was cloned. 181 of the 183 amino acids which overlap in the sequence derived from the cDNA and the proteolytic peptides were identical, and the two deviations are due to single base exchanges. A comparison of the amino acid sequence derived from the cloned cDNA with sequences of catalytic H(+)-ATPase subunits communicated by other laboratories revealed 98%, 96% and 94% identity with sequences from bovine adrenal medulla, from bovine kidney medulla and from clathrin-coated vesicles of bovine brain. Between 64% and 69% identity was obtained with sequences from fungi and plants. The data show that the catalytic subunit of V-ATPases is highly conserved during evolution. They indicate organ and species specificity in mammalians.     

Identification of nucleotide binding sites in V-type Na+-ATPase from Enterococcus hirae

A and B subunits of the V-type Na+-ATPase from Enterococcus hirae were suggested to possess nucleotide binding sites (Murata, T. et al., J. Biochem., 132, 789-794 (2002)), although the B subunit did not have the consensus sequence for nucleotide binding. To further characterize the binding sites in the V-ATPase, we did the photoaffinity labeling study using 8-azido-[alpha-32P]ATP. A and B subunits were labeled with 8-azido-[alpha-32P]ATP when analysed with SDS polyacrylamide gel electrophoresis. The peptide fragment of A subunit obtained by lysyl endopeptidase digestion after labeling showed a molecular size of 9 kDa and its amino acid sequencing revealed that it corresponded to residues Arg423-Lys494. The peptide fragment from B subunit after photoaffinity labeling and lysyl endopeptidase digestion showed the size of 5 kDa and corresponded to residues Phe404-Lys443. In our structure model, these peptides were close to the adenine ring of ATP. We suggest that non-catalytic B subunit of E. hirae V-ATPase has a nucleotide binding site, similarly to eukaryotic V-ATPases and F-ATPases.     

Cloning and sequencing of the gene encoding the large (alpha-) subunit of the proteasome from Thermoplasma acidophilum

The gene encoding the alpha-subunit of the proteasome from the archaebacterium Thermoplasma acidophilum was cloned and sequenced. The gene encodes for a polypeptide with 233 amino acid residues and a calculated molecular weight of 25870. Sequence similarity of the alpha-subunit with the Saccharomyces cerevisiae wild-type suppressor gene scll+ encoded polypeptide, which is probably identical with the subunit YC7-alpha of the yeast proteasome, lends support to a putative role of proteasomes in the regulation of gene expression. The significant sequence similarity to the various subunits of eukaryotic proteasomes make it likely that proteasomal proteins are encoded by one gene family of ancient origin.     

The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase

The gene encoding the proteolipid of the vacuolar H+-ATPase of yeast was cloned and sequenced. The deduced amino acid sequence of the yeast protein is highly homologous to that of the proteolipid from bovine chromaffin granules. In contrast to other membrane proteins the transmembrane segments of the bovine and yeast proteolipids were much more conserved than the hydrophilic parts. The fourth transmembrane segment, which contains the DCCD-binding site, was conserved 100%. Comparison of vacuolar and eubacterial proteolipids revealed a homology which pointed to a common ancestral gene that underwent gene duplication to form the vacuolar proteolipids. Additional support for this notion came from the amino acid sequences of subunits involved in the catalytic sectors of archaebacterial ATP synthase and plant and yeast vacuolar H+-ATPases, which reveal extensive sequence homology. Slight, but significant, homology between the archaebacterial and eubacterial ATP synthases was observed. These observations might suggest that the progenitor of ATP synthases was closely related to the present vacuolar H+-ATPases.     

Existence of two gamma subunits of the G proteins in brain

Although amino acid sequences have been determined for the alpha and beta subunits of Gs, Gi, and Go, sequences have not been reported for the gamma subunits of these G proteins. In the present paper, we determined the sequences of peptides prepared by partial proteolysis of two different forms of the gamma subunit of Gs, Gi, and Go from bovine brain. Using oligonucleotide probes based on the sequences of two of these peptides, a cDNA clone was isolated from a bovine adrenal cDNA library. This clone contained a 0.9-kilobase cDNA insert that included an open reading frame of 213 bases encoding a 71-amino acid polypeptide with an estimated Mr of 7850. The amino acid sequence predicted for the adrenal cDNA clone was identical to that determined for one form of the gamma subunit from brain. In addition, an antibody to a peptide based on the predicted amino acid sequence of this cDNA clone reacted specifically with one of the brain gamma subunits, indicating the adrenal cDNA clone encodes a gamma subunit present in both adrenal gland and brain. Also, evidence is presented, demonstrating the existence of a second, structurally distinct, form of the gamma subunit of Gs, Gi, and Go in brain.     

Type 1C fimbriae of a uropathogenic Escherichia coli strain: cloning and characterization of the genes involved in the expression of the 1C antigen and nucleotide sequence of the subunit gene

The genes responsible for expression of type 1C fimbriae have been cloned from the uropathogenic Escherichia coli strain AD110 in the plasmid vector pACYC184. Analysis of deletion mutants from these plasmids showed that a 7-kb DNA fragment was required for biosynthesis of 1C fimbriae. Further analysis of this DNA fragment showed that four genes are present encoding proteins of 16, 18.5, 21 and 89 kDal. A DNA fragment encoding the 16-kDal fimbrial subunit has been cloned. The nucleotide sequence of the structural gene and of the C- and N-terminal flanking regions was determined. The structural gene codes for a polypeptide of 181 amino acids, including a 24-residue N-terminal signal sequence. The nucleotide sequence and the deduced amino acid sequence of the 1C subunit gene were compared with the sequences of the fimA gene, encoding the type 1 fimbrial subunit of E. coli K-12. The data show absolute homology at the N- and C-termini; there is less, but significant homology in the region between the N- and C-termini. Comparison of the amino acid compositions of the 1C and FimA subunit proteins with those of the F72 and PapA proteins (subunits for P-fimbriae) revealed that homology between these two sets of fimbrial subunits is also maximal at the N- and C-termini.     

Unorthodox expression of an enzyme: evidence for an untranslated region within carA from Pseudomonas aeruginosa.

The genes encoding carbamoylphosphate synthetase from Pseudomonas aeruginosa PAO1 were cloned in Escherichia coli. Deletion and transposition analysis determined the locations of carA, encoding the small subunit, and carB, encoding the large subunit, on the chromosomal insert. The nucleotide sequence of carA and the flanking regions was determined. The derived amino acid sequence for the small subunit of carbamoylphosphate synthetase from P. aeruginosa exhibited 68% homology with its counterparts in E. coli and Salmonella typhimurium. The derived sequences in the three organisms were essentially identical in the three polypeptide segments that are conserved in glutamine amidotransferases but showed low homology at the amino- and carboxy-terminal regions. The amino-terminal amino acid sequences were determined for the large and small subunits. The first 15 amino acids of the large subunit were identical to those derived from the carB sequence. However, comparison of the derived sequence for carA with the amino-terminal amino acid sequence for the small subunit suggested that codons 5 to 8 are not translated. The DNA sequence for the region encompassing these four codons was confirmed by direct sequencing of chromosomal DNA after amplification by the polymerase chain reaction. The mRNA sequence was also deduced by in vitro synthesis of cDNA, enzymatic amplification, and sequencing, confirming that 12 nucleotides in the 5' terminal of carA are transcribed but are not translated.     

Molecular cloning and primary structure of human chromogranin A (secretory protein I) cDNA

Chromogranin A (CGA), also referred to as secretory protein I, is an acidic protein that has been detected in all neuroendocrine cell types examined and is often present in large amounts relative to other secreted proteins. For example, CGA comprises at least 40% of the soluble protein of the adrenal chromaffin granule, and it appears to be the major secretory protein in the parathyroid secretory granules. CGA complementary DNAs (cDNAs) from bovine adrenal and pituitary have recently been cloned and sequenced and found to be nearly identical. A region of bovine CGA has a high degree of amino acid sequence identity to pancreastatin, a recently isolated porcine peptide that inhibits glucose-induced insulin secretion. This suggests that CGA may be a prohormone. We have cloned and sequenced a human cDNA encoding CGA. This human CGA cDNA has an overall 86% nucleic acid identity to the bovine cDNA. Like the bovine CGA cDNA, the human cDNA has little homology to pancreastatin at the 5' region of this peptide but significant amino acid homology to the carboxyl-terminal portion of pancreastatin where the biologic activity resides. There is an area within the pancreastatin region of human CGA and porcine pancreastatin with a 70% amino acid identity to the calcium-binding moiety of the E-F hand proteins such as parvalbumin and oncomodulin. These data suggest that CGA and pancreastatin may both be members of a larger family of calcium-binding proteins.     

Sequence analysis of the large and small subunits of human ribonucleotide reductase.

We have isolated and sequenced overlapping cDNA clones from a breast carcinoma cDNA library containing the entire coding region of both the R1 and R2 subunits of the human ribonucleotide reductase gene. The coding region of the human R1 subunit comprises 2376 nucleotides and predicts a polypeptide of 792 amino acids (calculated molecular mass 90,081). The sequence of this subunit is almost identical to the equivalent mouse ribonucleotide reductase subunit with 97.7% homology between the mouse and human R1 subunit amino acid sequences. The coding region of the human R2 subunit of ribonucleotide reductase comprises 1170 nucleotides and predicts a polypeptide of 389 amino acids (calculated molecular mass 44,883), which is one amino acid shorter than the equivalent mouse subunit. The human and mouse R2 subunits display considerable homology in their carboxy-terminal amino acid sequences, with 96.3% homology downstream of amino acid 68 of the human and mouse R2 proteins. However, the amino-terminal portions of these two proteins are more divergent in sequence, with only 69.2% homology in the first 68 amino acids.     

Cloning and nucleotide sequence analysis of the serotype 2 fimbrial subunit gene of Bordetella pertussis

An oligonucleotide probe complementary to the beginning of the gene encoding the serotype 2(ST2) fimbrial subunit of Bordetella pertussis was synthesized and a cloned DNA fragment hybridizing with the probe identified and sequenced. Several lines of evidence indicate that an open reading frame with coding information for a polypeptide of 207 amino acids, including a 26-amino-acid signal sequence, is the ST2 gene. The protein deduced from the nucleotide sequence shows good agreement with the NH2-terminal amino acid sequence, amino acid composition and molecular weight of the purified fimbrial subunit. In addition, the proposed ST2 subunit is shown to have homology with other fimbrial subunits.     

The amino-terminal domain of the E subunit of vacuolar H(+)-ATPase (V-ATPase) interacts with the H subunit and is required for V-ATPase function

Vacuolar H(+)-ATPases (V-ATPases) are highly conserved proton pumps that couple hydrolysis of cytosolic ATP to proton transport out of the cytosol. Although it is generally believed that V-ATPases transport protons by a rotary catalytic mechanism analogous to that used by F(1)F(0)-ATPases, the structure and subunit composition of the central or peripheral stalk of the multisubunit complex are not well understood. We searched for proteins that bind to the E subunit of V-ATPase using the yeast two-hybrid assay and identified the H subunit as an interacting partner. Physical association between the E and H subunits of V-ATPase was confirmed in vitro by precipitation assays. Deletion mapping analysis revealed that a 78-amino acid fragment at the amino terminus of the E subunit was sufficient for binding to the H subunit. Expression of the amino-terminal fragments of the E subunits from human and yeast as dominant-negative mutants resulted in dramatic decreases in bafilomycin A(1)-sensitive ATP hydrolysis and proton transport activities of V-ATPase. Our data demonstrate the physiological significance of the interaction between the E and H subunits of V-ATPase and extend previous studies on the arrangement of subunits on the peripheral stalk of V-ATPase.     

Subunit C of the Vacuolar-Type ATPase from the Vanadium-Rich Ascidian Ascidia sydneiensis samea Rescued the pH Sensitivity of Yeast vma5 Mutants

A vanadium-accumulating ascidian, Ascidia sydneiensis samea, expresses vacuolar-type H⁺-ATPases (V-ATPases) on the vacuole membrane of the vanadium-containing blood cells known as vanadocytes. Previously, we showed that the contents of their vacuoles are extremely acidic and that a V-ATPase-specific inhibitor, bafilomycin A₁, neutralized the contents of the vacuoles. To understand the function of V-ATPase in vanadocytes, we isolated complementary DNA encoding subunit C of V-ATPase from vanadocytes because this subunit has been known to be responsible for the assembly of V-ATPases and to regulate the ATPase activity of V-ATPases. The cloned cDNA was 1443 nucleotides in length, and encoded a putative 384 amino acid protein. By expressing the ascidian cDNA for subunit C under the control of a galactose-inducible promoter, the pH-sensitive phenotype of the corresponding vma5 mutant of a budding yeast was rescued. This result showed that the ascidian cDNA for subunit C functioned in yeast cells. Received August 11, 2000; accepted March 5, 2001.     

Isolation and sequence of rat testis cDNA for a calcium binding polypeptide similar to the regulatory subunit of calcineurin.

We have cloned and sequenced rat testis cDNAs coding for a calcium binding polypeptide similar to calcineurin beta subunit, the Ca(2+)-binding subunit of the Ca2+/calmodulin stimulated protein phosphatase. Rat testis cDNA library was screened with a monoclonal antibody Va1 raised against bovine brain calcineurin beta subunit. The deduced amino acid sequence is similar to that of human brain calcineurin beta subunit with respect to containing four putative calcium binding sites. However, distinct differences were found: 1) The cloned cDNA had six amino acids polypeptide tail at carboxy-terminal which is absent in human brain calcineurin beta subunit. This amino acids tail makes the carboxy-terminal highly hydrophilic in contrast to the human brain beta subunit which is hydrophobic at carboxy-terminal; 2) eleven amino acids at the N terminal of the cloned cDNA were completely different from the corresponding region of the brain calcineurin beta subunit.     

水稻液泡ATPase B 亚基基因的克隆及其在低磷胁迫下的表达特征分析

利用抑制性扣除杂交(SSH)技术构建水稻(Oryza sativa L.)根系磷饥饿诱导cDNA文库,获得编码液泡ATPase (V-ATPase) B亚基的克隆,通过反转录PCR方法获得该基因的完整序列.该基因编码487个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量为54.06 kD,等电点为4.99.Southern印迹表明,V-ATPase B亚基基因在水稻基因组中以单拷贝形式存在.氨基酸同源性分析发现,V-ATPase B亚基是一个较为保守的蛋白亚基,其序列变化伴随生物的进化过程同步进行.Northern印迹表明,V-ATPase B亚基在水稻根系中受到磷饥饿诱导表达,磷饥饿6～12 h出现表达高峰,而在叶片中表达高峰有所滞后(24～48 h).在缺磷环境条件下,ATPase B亚基可能通过提高其表达量,进而提高质子转运活性,形成跨膜的电化学梯度,为体内储备磷跨液泡膜运输提供能量,从而提高植物体内磷的利用效率及其耐低磷的能力.     

Sequencing of full-length cDNA encoding the alpha and beta subunits of human casein kinase II from human platelets and megakaryocytic cells. Expression of the casein kinase IIalpha intronless gene in a megakaryocytic cell line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --> His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets.     

桔小实蝇V-ATPase G亚基基因的克隆及组织表达特异性分析

空泡型ATP酶（vacuolar-type H⁺-ATPase, V-ATPase）作为质子泵几乎在所有的真核生物细胞中发挥重要作用。本研究利用RT-PCR和RACE技术获得了桔小实蝇Bactrocera dorsalis (Hendel)V-ATPase G亚基序列全长, 命名为BdorATPG。测序结果表明, BdorATPG阅读框全长354 bp, 编码117个氨基酸。氨基酸序列比对表明, BdorATPG的N端序列与其他物种的ATPG亚基对应区域具有较高的序列一致性。BdorATPG与拟暗果蝇Drosophila pseudoobscura ATPG亚基的氨基酸序列一致性最高, 为88.9%。三维结构模建结果表明, BdorATPG N端（第1~59位氨基酸）序列为α-螺旋结构, 亲水性和疏水性氨基酸在螺旋两侧呈对称分布。BdorATPG在不同组织中的荧光定量PCR分析表明, BdorATPG在各组织中都有表达, 其中在触角中的表达量最高; 在雄虫生殖节中的表达量是雌虫中的6.04倍。结果提示BdorATPG可能在雄虫生殖生理过程中发挥重要作用。