Regulation of AE2-mediated Cl− Transport by Intracellular or by Extracellular pH Requires Highly Conserved Amino Acid Residues of the AE2 NH2-terminal Cytoplasmic Domain

We reported recently that regulation by intracellular pH (pHi) of the murine Cl−/HCO3− exchanger AE2 requires amino acid residues 310–347 of the polypeptide's NH2-terminal cytoplasmic domain. We have now identified individual amino acid residues within this region whose integrity is required for regulation of AE2 by pH. 36Cl− efflux from AE2-expressing Xenopus oocytes was monitored during variation of extracellular pH (pHo) with unclamped or clamped pHi, or during variation of pHi at constant pHo. Wild-type AE2–mediated 36Cl− efflux was profoundly inhibited by acid pHo, with a value of pHo(50) = 6.87 ± 0.05, and was stimulated up to 10-fold by the intracellular alkalinization produced by bath removal of the preequilibrated weak acid, butyrate. Systematic hexa-alanine [(A)6]bloc substitutions between aa 312–347 identified the greatest acid shift in pHo(50) value, ∼0.8 pH units in the mutant (A)6342–347, but only a modest acid-shift in the mutant (A)6336–341. Two of the six (A)6 mutants retained normal pHi sensitivity of 36Cl− efflux, whereas the (A)6 mutants 318–323, 336–341, and 342–347 were not stimulated by intracellular alkalinization. We further evaluated the highly conserved region between aa 336–347 by alanine scan and other mutagenesis of single residues. Significant changes in AE2 sensitivity to pHo and to pHi were found independently and in concert. The E346A mutation acid-shifted the pHo(50) value to the same extent whether pHi was unclamped or held constant during variation of pHo. Alanine substitution of the corresponding glutamate residues in the cytoplasmic domains of related AE anion exchanger polypeptides confirmed the general importance of these residues in regulation of anion exchange by pH. Conserved, individual amino acid residues of the AE2 cytoplasmic domain contribute to independent regulation of anion exchange activity by pHo as well as pHi.

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2P-041 Roles of individual amino acid residues in folding/unfolding kinetics of lattice proteins studied with a simple statistical mechanical model(Protein:Property,The 47th Annual Meeting of the Biophysical Society of Japan)

Seibutsu Butsuri ◽

10.2142/biophys.49.s113_1 ◽

2009 ◽

Vol 49 (supplement) ◽

pp. S113

Author(s):

Haruo Abe ◽

Hiroshi Wako

Keyword(s):

Amino Acid ◽

Mechanical Model ◽

Individual Amino Acid ◽

Amino Acid Residues ◽

Biophysical Society ◽

Statistical Mechanical Model ◽

Statistical Mechanical ◽

Lattice Proteins ◽

Unfolding Kinetics ◽

Kinetics Of

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Amino Acid Residues in the Cytoplasmic Domain of the Mason-Pfizer Monkey Virus Glycoprotein Critical for Its Incorporation into Virions

Journal of Virology ◽

10.1128/jvi.79.18.11559-11568.2005 ◽

2005 ◽

Vol 79 (18) ◽

pp. 11559-11568 ◽

Cited By ~ 8

Author(s):

Chisu Song ◽

Keith Micoli ◽

Helena Bauerova ◽

Iva Pichova ◽

Eric Hunter

Keyword(s):

Amino Acid ◽

Transmembrane Protein ◽

Critical Role ◽

Cytoplasmic Domain ◽

Virus Infectivity ◽

Amino Acid Residues ◽

Glycoprotein Complex ◽

Uptake Studies ◽

Domain Block ◽

And Function

ABSTRACT Assembly of an infectious retrovirus requires the incorporation of the envelope glycoprotein complex during the process of particle budding. We have recently demonstrated that amino acid substitutions of a tyrosine residue in the cytoplasmic domain block glycoprotein incorporation into budding Mason-Pfizer monkey virus (M-PMV) particles and abrogate infectivity (C. Song, S. R. Dubay, and E. Hunter, J. Virol. 77:5192-5200, 2003). To investigate the contribution of other amino acids in the cytoplasmic domain to the process of glycoprotein incorporation, we introduced alanine-scanning mutations into this region of the transmembrane protein. The effects of the mutations on glycoprotein biosynthesis and function, as well as on virus infectivity, have been examined. Mutation of two cytoplasmic residues, valine 20 and histidine 21, inhibits viral protease-mediated cleavage of the cytoplasmic domain that is observed during virion maturation, but the mutant virions show only moderately reduced infectivity. We also demonstrate that the cytoplasmic domain of the M-PMV contains three amino acid residues that are absolutely essential for incorporation of glycoprotein into virions. In addition to the previously identified tyrosine at residue 22, an isoleucine at position 18 and a leucine at position 25 each mediate the process of incorporation and efficient release of virions. While isoleucine 18 may be involved in direct interactions with immature capsids, antibody uptake studies showed that leucine 25 and tyrosine 22 are part of an efficient internalization signal in the cytoplasmic domain of the M-PMV glycoprotein. These results demonstrate that the cytoplasmic domain of M-PMV Env, in part through its YXXL-mediated endocytosis and intracellular trafficking signals, plays a critical role in the incorporation of glycoprotein into virions.

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