The vacuolar proton pump of Dictyostelium discoideum: molecular cloning and analysis of the 100 kDa subunit

1996 ◽  
Vol 109 (5) ◽  
pp. 1041-1051 ◽  
Author(s):  
T. Liu ◽  
M. Clarke
Keyword(s):  
Dictyostelium Discoideum ◽  
Proton Pump ◽  
Single Gene ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Contractile Vacuole ◽  
Proton Pumps ◽  
Variable Regions ◽  
Endosomal Membranes ◽  
Vacuolar Proton Pump

The vacuolar proton pump is a highly-conserved multimeric enzyme that catalyzes the translocation of protons across the membranes of eukaryotic cells. Its largest subunit (95-116 kDa) occurs in tissue and organelle-specific isoforms and thus may be involved in targeting the enzyme or modulating its function. In amoebae of Dictyostelium discoideum, proton pumps with a 100 kDa subunit are found in membranes of the contractile vacuole complex, an osmoregulatory organelle. We cloned the cDNA that encodes this 100 kDa protein and found that its sequence predicts a protein 45% identical (68% similar) to the corresponding mammalian proton pump subunit. Like the mammalian protein, the predicted Dictyostelium sequence contains six possible transmembrane domains and a single consensus sequence for N-linked glycosylation. Southern blot analysis detected only a single gene, which was designated vatM. Using genomic DNA and degenerate oligonucleotides based on conserved regions of the protein as primers, we generated products by polymerase chain reaction that included highly variable regions of this protein family. The cloned products were identical in nucleotide sequence to vatM, arguing that Dictyostelium cells contain only a single isoform of this proton pump subunit. Consistent with this interpretation, the amino acid sequences of peptides derived from a protein associated with endosomal membranes (Adessu et al. (1995) J. Cell Sci. 108, 3331–3337) match the predicted sequence of the protein encoded by vatM. Thus, a single isoform of the 100 kDa proton pump subunit appears to serve in both the contractile vacuole system and the endosomal/lysosomal system of Dictyostelium, arguing that this subunit is not responsible for regulating the differing abundance and function of proton pumps in these two compartments. Gene targeting experiments suggest that this subunit plays important (possibly essential) roles in Dictyostelium cells.

An immunocytochemical analysis of the vacuolar proton pump in Dictyostelium discoideum

1993 ◽  
Vol 105 (3) ◽  
pp. 849-859 ◽  
Author(s):  
K.V. Nolta ◽  
H. Padh ◽  
T.L. Steck
Keyword(s):  
Dictyostelium Discoideum ◽  
Proton Pump ◽  
Mammalian Cells ◽  
Proton Pumps ◽  
Multivesicular Bodies ◽  
Animal Cells ◽  
Intact Cells ◽  
Immunocytochemical Analysis ◽  
F1 Atpase ◽  
Vacuolar Proton Pump

Antisera were generated in rabbits against the vacuolar proton pump (V-H(+)-ATPase) purified from Dictyostelium discoideum. The antisera inhibited V-H(+)-ATPase but not F1-ATPase activity and immunoprecipitated and immunoblotted only the polypeptide subunits of the V-H(+)-ATPase from cell homogenates. Immunocytochemical analysis of intact cells and subcellular fractions showed that the predominant immunoreactive organelles were clusters of empty, irregular vacuoles of various sizes and shapes, which corresponded to the acidosomes. The cytoplasmic surfaces of lysosomes, phagosomes and the tubular spongiome of the contractile vacuole also bore the pump antigen. The lumina of multivesicular bodies were often stained intensely; the internalized antigen may have been derived from acidosomes by autophagy. Antibodies against V-H(+)-ATPases from plant and animal cells cross-reacted with the proton pumps of Dictyostelium. Antisera directed against the V-H(+)-ATPase of Dictyostelium decorated a profusion of small vacuoles scattered throughout the cytoplasm of hepatocytes, epithelial cells, macrophages and fibroblasts. The pattern paralleled that of the endocytic and acidic spaces; there was no clear indication of discrete acidosomes in these mammalian cells. We conclude that the V-H(+)-ATPase in Dictyostelium is distributed among diverse endomembrane organelles and is immunologically cross-reactive with the proton pumps on endocytic vacuoles in mammalian cells.

scholarly journals Regulatory assembly of the vacuolar proton pump V o V 1 -ATPase in yeast cells by FLIM-FRET

2010 ◽  
Author(s):  
Stefan Ernst ◽  
Claire Batisse ◽  
Nawid Zarrabi ◽  
Bettina Böttcher ◽  
Michael Börsch
Keyword(s):  
Proton Pump ◽  
Yeast Cells ◽  
Vacuolar Proton Pump

Cloning and expression of a cDNA that comprises part of the gene coding for a spore coat protein of Dictyostelium discoideum

1984 ◽  
Vol 4 (11) ◽  
pp. 2273-2278
Author(s):  
B C Dowds ◽  
W F Loomis
Keyword(s):  
Dictyostelium Discoideum ◽  
Cdna Clone ◽  
Single Gene ◽  
Polyclonal Antiserum ◽  
Cloning And Expression ◽  
Spore Coat ◽  
Coat Proteins ◽  
Developmentally Regulated ◽  
Gene Coding ◽  
Spore Coat Proteins

The three major spore coat proteins of Dictyostelium discoideum are developmentally regulated, cell-type-specific proteins. They are packaged in prespore vesicles and then secreted to form the outer layer of spore coats. We have isolated a cDNA clone from the gene coding for one of these proteins, SP96, a glycoprotein of 96,000 daltons. We screened the cDNA bank by the method of hybrid select translation followed by immunoprecipitation of the translation products with SP96-specific polyclonal antiserum. We found that the gene was first transcribed into stable mRNA a few hours before the time of detection of SP96 synthesis and that the mRNA, like the protein, accumulated specifically in prespore cells and spores. SP96 constituted the same proportion of newly synthesized protein as the proportion of its message in polyadenylated RNA. SP96 appeared to be encoded by a single gene as judged by Southern blot analysis of digested genomic DNA hybridized to the cDNA clone.

The COOH termini of NBC3 and the 56-kDa H+-ATPase subunit are PDZ motifs involved in their interaction

2003 ◽  
Vol 284 (3) ◽  
pp. C667-C673 ◽  
Author(s):  
Alexander Pushkin ◽  
Natalia Abuladze ◽  
Debra Newman ◽  
Vladimir Muronets ◽  
Pejvak Sassani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Proton Pump ◽  
Rat Kidney ◽  
Pdz Domain ◽  
Intercalated Cells ◽  
Regulatory Factor ◽  
Atpase Subunit ◽  
Sodium Bicarbonate Cotransporter ◽  
Terminal Amino ◽  
Vacuolar Proton Pump

The electroneutral sodium bicarbonate cotransporter 3 (NBC3) coimmunoprecipitates from renal lysates with the vacuolar H+-ATPase. In renal type A and B intercalated cells, NBC3 colocalizes with the vacuolar H+-ATPase. The involvement of the COOH termini of NBC3 and the 56-kDa subunit of the proton pump in the interaction of these proteins was investigated. The intact and modified COOH termini of NBC3 and the 56-kDa subunit of the proton pump were synthesized, coupled to Sepharose beads, and used to pull down kidney membrane proteins. Both the 56- and the 70-kDa subunits of the proton pump, as well as a PDZ domain containing protein Na+/H+ exchanger regulatory factor 1 (NHERF-1), were bound to the intact 18 amino acid NBC3 COOH terminus. A peptide truncated by five COOH-terminal amino acids did not bind these proteins. Replacement of the COOH-terminal leucine with glycine blocked binding of both the proton pump subunits but did not affect binding of NHERF-1. The 18 amino acid COOH terminus of the 56-kDa subunit of the proton pump bound NHERF-1 and NBC3, but the truncated and modified peptide did not. A complex of NBC3, the 56-kDa subunit of the proton pump, and NHERF-1 was identified in rat kidney. The data indicate that the COOH termini of NBC3 and the 56-kDa subunit of the vacuolar proton pump are PDZ-interacting motifs that are necessary for the interaction of these proteins. NHERF-1 is involved in the interaction of NBC3 and the vacuolar proton pump.

scholarly journals Modified Rhodopsins From Aureobasidium pullulans Excel With Very High Proton-Transport Rates

2021 ◽  
Vol 8 ◽  
Author(s):  
Sabine Panzer ◽  
Chong Zhang ◽  
Tilen Konte ◽  
Celine Bräuer ◽  
Anne Diemar ◽  
...  
Keyword(s):  
Proton Pump ◽  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Aureobasidium Pullulans ◽  
Green Light ◽  
Maximal Activity ◽  
Site Directed Mutagenesis ◽  
Proton Pumps ◽  
C Terminus ◽  
Pump Activity

Aureobasidium pullulans is a black fungus that can adapt to various stressful conditions like hypersaline, acidic, and alkaline environments. The genome of A. pullulans exhibits three genes coding for putative opsins ApOps1, ApOps2, and ApOps3. We heterologously expressed these genes in mammalian cells and Xenopus oocytes. Localization in the plasma membrane was greatly improved by introducing additional membrane trafficking signals at the N-terminus and the C-terminus. In patch-clamp and two-electrode-voltage clamp experiments, all three proteins showed proton pump activity with maximal activity in green light. Among them, ApOps2 exhibited the most pronounced proton pump activity with current amplitudes occasionally extending 10 pA/pF at 0 mV. Proton pump activity was further supported in the presence of extracellular weak organic acids. Furthermore, we used site-directed mutagenesis to reshape protein functions and thereby implemented light-gated proton channels. We discuss the difference to other well-known proton pumps and the potential of these rhodopsins for optogenetic applications.

scholarly journals Effects of inhibitors of the vacuolar proton pump on hepatic heterophagy and autophagy

2001 ◽  
Vol 1510 (1-2) ◽  
pp. 243-257 ◽  
Author(s):  
Seyed Ali Mousavi ◽  
Rune Kjeken ◽  
Trond Olav Berg ◽  
Per Ottar Seglen ◽  
Trond Berg ◽  
...  
Keyword(s):  
Proton Pump ◽  
Vacuolar Proton Pump

Isolation and characterization of some novel genes of the apolipoprotein A-I family in Japanese eel, Anguilla japonica

2011 ◽  
Vol 6 (4) ◽  
pp. 545-557 ◽  
Author(s):  
Malay Choudhury ◽  
Takahiro Oku ◽  
Shoji Yamada ◽  
Masaharu Komatsu ◽  
Keita Kudoh ◽  
...  
Keyword(s):  
Amino Acids ◽  
Molecular Weight ◽  
Amino Acid ◽  
Consensus Sequence ◽  
Structural Features ◽  
Lipid Binding ◽  
Japanese Eel ◽  
Amino Acid Sequences ◽  
Novel Genes ◽  
Isolation And Characterization

AbstractApolipoproteins such as apolipoprotein (apo) A-I, apoA-IV, and apoE are lipid binding proteins synthesized mainly in the liver and the intestine and play an important role in the transfer of exogenous or endogenous lipids through the circulatory system. To investigate the mechanism of lipid transport in fish, we have isolated some novel genes of the apoA-I family, apoIA-I (apoA-I isoform) 1–11, from Japanese eel by PCR amplification. Some of the isolated genes of apoIA-I corresponded to 28kDa-1 cDNAs which had already been deposited into the database and encoded an apolipoprotein with molecular weight of 28 kDa in the LDL, whereas others seemed to be novel genes. The structural organization of all apoIA-Is consisted of four exons separated by three introns. ApoIA-I10 had a total length of 3232 bp, whereas other genes except for apoIA-I9 ranged from 1280 to 1441 bp. The sequences of apoIA-Is at the exon-intron junctions were mostly consistent with the consensus sequence (GT/AG) at exon-intron boundaries, whereas the sequences of 3′ splice acceptor in intron 1 of apoIA-I1-7 were (AC) but not (AG). The deduced amino acid sequences of all apoIA-Is contained a putative signal peptide and a propeptide of 17 and 5 amino acid residues, respectively. The mature proteins of apoIA-I1-3, 7, and 8 consisted of 237 amino acids, whereas those of apoIA-I4-6 consisted of 239 amino acids. The mature apoIA-I10 sequence showed 65% identity to amino acid sequence of apoIA-I11 which was associated with an apolipoprotein with molecular weight of 23 kDa in the VLDL. All these mature apoIA-I sequences satisfied the common structural features depicted for the exchangeable apolipoproteins such as apoA-I, apoA-IV, and apoE but apoIA-I11 lacked internal repeats 7, 8, and 9 when compared with other members of apoA-I family. Phylogenetic analysis showed that these novel apoIA-Is isolated from Japanese eel were much closer to apoA-I than apoA-IV and apoE, suggesting new members of the apoA-I family.

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