Characterization of a Histidine Rich Cluster of Amino Acids in the Cytoplasmic Domain of the Na+/H+ Exchanger

2000 ◽  
Vol 20 (3) ◽  
pp. 185-197 ◽  
Author(s):  
Pavel Dibrov ◽  
Rakhilya Murtazina ◽  
James Kinsella ◽  
Larry Fliegel
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Cytoplasmic Domain ◽  
Intact Protein ◽  
Affinity Column ◽  
Rich Cluster ◽  
Protein Mutation ◽  
Conserved Sequence ◽  
Carboxyl Terminal

We examined the function of a highly conserved Histidine rich sequence ofamino acids found in the carboxyl-terminal of the Na+/H+exchanger (NHE1). A fusion protein containing the sequenceHYGHHH (540–545) and the balance of the carboxyl terminalof the protein did not bind calcium but bound to an immobilizedmetal affinity column and could be used to partially purify theexchanger protein. Mutation of the sequence to either HYGAAA orHYGRRR did not affect activity of the intact protein. Mutationto HHHHHH did not affect proton activation of the Na+/H+exchanger or localization but caused a decreased maximal velocitysuggesting that this conserved sequence is important in maximalactivity of the Na+/H+ exchanger.

Expression, purification, and characterization of the carboxyl-terminal region of the Na+/H+ exchanger

1998 ◽  
Vol 76 (5) ◽  
pp. 837-842 ◽  
Author(s):  
Daniel Gebreselassie ◽  
Krishna Rajarathnam ◽  
Larry Fliegel
Keyword(s):  
Circular Dichroism ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Gel Electrophoresis ◽  
Inclusion Bodies ◽  
Cytoplasmic Domain ◽  
Random Coil ◽  
Glutathione S Transferase ◽  
Carboxyl Terminal ◽  
Circular Dichroïsm

The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.Key words: Na+/H+ exchanger, pH regulation, membrane protein, circular dichroism.

scholarly journals Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter

1988 ◽  
Vol 8 (3) ◽  
pp. 1327-1335
Author(s):  
J V Cox ◽  
E Lazarides
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Transporter Protein ◽  
Isolation And Characterization ◽  
Anion Transporter ◽  
Primary Structures ◽  
Membrane Spanning

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

scholarly journals Characterization of a Di-leucine–based Signal in the Cytoplasmic Tail of the Nucleotide-pyrophosphatase NPP1 That Mediates Basolateral Targeting but not Endocytosis

2001 ◽  
Vol 12 (10) ◽  
pp. 3004-3015 ◽  
Author(s):  
Valérie Bello ◽  
James W. Goding ◽  
Vicki Greengrass ◽  
Adnan Sali ◽  
Valentina Dubljevic ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cytoplasmic Tail ◽  
Full Length ◽  
Apical Surface ◽  
Short Sequence ◽  
Conserved Sequence ◽  
Nucleotide Pyrophosphatase ◽  
Targeting Signal ◽  
Basolateral Targeting

Enzymes of the nucleotide pyrophosphatase/phosphodiesterase (NPPase) family are expressed at opposite surfaces in polarized epithelial cells. We investigated the targeting signal of NPP1, which is exclusively expressed at the basolateral surface. Full-length NPP1 and different constructs and mutants were transfected into the polarized MDCK cell line. Expression of the proteins was analyzed by confocal microscopy and surface biotinylation. The basolateral signal of NPP1 was identified as a di-leucine motif located in the cytoplasmic tail. Mutation of either or both leucines largely redirected NPP1 to the apical surface. Furthermore, addition of the conserved sequence AAASLLAP redirected the apical nucleotide pyrophosphatase/phosphodiesterase NPP3 to the basolateral surface. Full-length NPP1 was not significantly internalized. However, when the cytoplasmic tail was deleted upstream the di-leucine motif or when the six upstream flanking amino acids were deleted, the protein was mainly found intracellularly. Endocytosis experiments indicated that these mutants were endocytosed from the basolateral surface. These results identify the basolateral signal of NPP1 as a short sequence including a di-leucine motif that is dominant over apical determinants and point to the importance of surrounding amino acids in determining whether the signal will function as a basolateral signal only or as an endocytotic signal as well.

scholarly journals Construction and Evaluation of a Novel Bifunctional N-Carbamylase–d-Hydantoinase Fusion Enzyme

2000 ◽  
Vol 66 (5) ◽  
pp. 2133-2138 ◽  
Author(s):  
Geun-Joong Kim ◽  
Dong-Eun Lee ◽  
Hak-Sung Kim
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Bacillus Stearothermophilus ◽  
Gel Filtration ◽  
Intact Protein ◽  
E Coli ◽  
Fusion Enzyme ◽  
N Terminus ◽  
Fusion Enzymes ◽  
Hydantoin Derivatives

ABSTRACT A fully enzymatic process employing two sequential enzymes,d-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity for the production ofd-amino acids. To test the possibility of generating a bifunctional fusion enzyme, we constructed a fusion protein via end-to-end fusion of a whole gene that encodes an intact protein at the N terminus of the d-hydantoinase. Firstly, maltose-binding protein (MBP) gene of E. coli was fused withd-hydantoinase gene from Bacillus stearothermophilus SD1, and the properties of the resulting fusion protein (MBP-HYD) were compared with those of natived-hydantoinase. Gel filtration and kinetic analyses clearly demonstrated that the typical characteristics ofd-hydantoinase are maintained even in a fusion state. Based on this result, we constructed an artificial fusion enzyme composed of the whole length of N-carbamylase (304 amino acids [aa]) from Agrobacterim radiobacter NRRL B11291 andd-hydantoinase (471 aa). The fusion enzyme (CAB-HYD) was functionally expressed with an expected molecular mass of 86 kDa and efficiently converted exogenous hydantoin derivatives to thed-amino acids. A related d-hydantoinase (HYD1) gene from Bacillus thermocatenulatus GH2 was also fused with the N-carbamylase gene at its N terminus. The resulting enzyme (CAB-HYD1) was bifunctional as expected and showed better performance than the CAB-HYD fusion enzyme. The conversion of hydantoin derivatives to corresponding amino acids by the fusion enzymes was much higher than that by the separately expressed enzymes, and comparable to that by the coexpressed enzymes. Thus, the fusion enzyme might be useful as a potential biocatalyst for the production of nonnatural amino acids.

scholarly journals Alternative primary structures in the transmembrane domain of the chicken erythroid anion transporter.

1988 ◽  
Vol 8 (3) ◽  
pp. 1327-1335 ◽  
Author(s):  
J V Cox ◽  
E Lazarides
Keyword(s):  
Amino Acids ◽  
Transmembrane Domain ◽  
Cytoplasmic Domain ◽  
Band 3 ◽  
Transporter Protein ◽  
Isolation And Characterization ◽  
Anion Transporter ◽  
Primary Structures ◽  
Membrane Spanning

Isolation and characterization of the chicken erythroid anion transporter (band 3) cDNA clone, pCHB3-1, revealed that the chicken erythroid band 3 polypeptide is 844 amino acids in length with a predicted mass of 109,000 daltons. This polypeptide is composed of a hydrophilic N-terminal cytoplasmic domain and a hydrophobic C-terminal transmembrane domain. The approximately 90 N-terminal amino acids of the human and murine erythroid band 3 polypeptides are absent in the predicted sequence of the chicken erythroid band 3 polypeptide. The absence of this very acidic N-terminal region is consistent with the lack of binding of glyceraldehyde-3-phosphate dehydrogenase to chicken erythroid band 3, as well as the relatively basic isoelectric point observed for this molecule. The remainder of the cytoplasmic domain shows little similarity to the cytoplasmic domain of the murine and human erythroid band 3, with the exception of the putative ankyrin-binding site, which is highly conserved. In contrast, the transmembrane domain of the chicken band 3 polypeptide is very similar to that of the murine erythroid and human nonerythroid band 3 polypeptides. The transmembrane domain contains 10 hydrophobic regions that could potentially traverse the membrane 12 to 14 times. In addition, a variant of chicken erythroid band 3, pCHB3-2, was cloned in which one of the hydrophobic regions of pCHB3-1 is lacking. The transcript complementary to pCHB3-2 accumulated in chicken erythroid cells in a similar manner as the transcript complementary to pCHB3-1 during embryonic development. This is the first example of a transporter protein or ion channel with alternative primary structures in its membrane-spanning segments.

Isolation and Characterization of the Cryptococcus neoformans MATa Pheromone Gene

Genetics ◽  
2002 ◽  
Vol 160 (3) ◽  
pp. 935-947
Author(s):  
Carol M McClelland ◽  
Jianmin Fu ◽  
Gay L Woodlee ◽  
Tara S Seymour ◽  
Brian L Wickes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Cryptococcus Neoformans ◽  
Mating Types ◽  
Filament Formation ◽  
Gene Encoding ◽  
Conserved Sequence ◽  
Isolation And Characterization ◽  
Conserved Genes

Abstract Cryptococcus neoformans is a heterothallic basidiomycete with two mating types, MATa and MATα. The mating pathway of this fungus has a number of conserved genes, including a MATα-specific pheromone (MFα1). A modified differential display strategy was used to identify a gene encoding the MATa pheromone. The gene, designated MFa1, is 42 amino acids in length and contains a conserved farnesylation motif. MFa1 is present in three linked copies that span a 20-kb fragment of MATa-specific DNA and maps to the MAT-containing chromosome. Transformation studies showed that MFa1 induced filament formation only in MATα cells, demonstrating that MFa1 is functionally conserved. Sequence analysis of the predicted Mfa1 and Mfα1 proteins revealed that, in contrast to other fungi such as Saccharomyces cerevisiae, the C. neoformans pheromone genes are structurally and functionally conserved. However, unlike the MFα1 gene, which is found in MATα strains of both varieties of C. neoformans, MFa1 is specific for the neoformans variety of C. neoformans.

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