Identification of a domain in the alpha-subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the gamma-subunit.
The oxaloacetate decarboxylase Na+ pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral alpha-subunit associated with two integral membrane-bound subunits (beta and gamma). The alpha-subunit contains the carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The gamma-subunit plays a profound role in the assembly of the complex. It interacts with the beta-subunit through its N-terminal membrane-spanning region and with the alpha-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 alpha-2 and the C-terminal domain of gamma-2, termed gamma'-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 alpha-2 was identified by deletion mutagenesis to be essential for the binding with gamma'-2. This portion of the alpha-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 gamma'-2. This peptide therefore represents a newly identified, separate domain of the alpha-subunit and has been called the 'association domain'.
Dahinden
Pius
P
Institute of Microbiology ETH Zürich, ETH Hönggerberg, Zürich, Switzerland.
Pos
Klaas M
KM
Dimroth
Peter
P
D000595:Amino Acid Sequence; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002846:Chromatography, Affinity; D004274:DNA, Recombinant; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D004274:DNA, Recombinant; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase