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Identification of a domain in the α‐subunit of the oxaloacetate decarboxylase Na + pump that accomplishes complex formation with the γ‐subunit
Author(s) -
Dahinden Pius,
Pos Klaas M.,
Dimroth Peter
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2004.04524.x
Subject(s) - protein subunit , specificity factor , gamma subunit , g alpha subunit , biology , interleukin 10 receptor, alpha subunit , biochemistry , peptide sequence , n terminus , mutagenesis , gamma aminobutyric acid receptor subunit alpha 1 , escherichia coli , mutation , gene , rna polymerase
The oxaloacetate decarboxylase Na + pumps OAD‐1 and OAD‐2 of Vibrio cholerae are composed of a peripheral α‐subunit associated with two integral membrane‐bound subunits (β and γ). The α‐subunit contains the carboxyltransferase domain in its N‐terminal portion and the biotin‐binding domain in its C‐terminal portion. The γ‐subunit plays a profound role in the assembly of the complex. It interacts with the β‐subunit through its N‐terminal membrane‐spanning region and with the α‐subunit through its hydrophilic C‐terminal domain. The biochemical and structural requirements for the latter interaction were analysed with OAD‐2 expression clones for subunit α‐2 and the C‐terminal domain of γ‐2, termed γ′‐2. If the two proteins were synthesized together in Escherichia coli they formed a complex that was stable at neutral pH and dissociated at pH<5.0. An internal stretch of 40 amino acids of α‐2 was identified by deletion mutagenesis to be essential for the binding with γ′‐2. This portion of the α‐subunit is connected via flexible linker peptides to the carboxyltransferase domain at its N terminus and to the biotin‐binding domain at its C terminus. Results of site‐directed mutagenesis indicated that a conserved tyrosine (491) and threonine 494 of this peptide contributed significantly to the stability of the complex with γ′‐2. This peptide therefore represents a newly identified, separate domain of the α‐subunit and has been called the ‘association domain’.