tailieunhanh - Báo cáo khoa học: Identification of a domain in the a-subunit of the oxaloacetate decarboxylase Na+ pump that accomplishes complex formation with the c-subunit

The oxaloacetate decarboxylase Na + pumps OAD-1 and OAD-2 ofVibrio choleraeare composed of a peripherala-subunit associated with two integ-ral membrane-bound subunits (bandc). The a-subunit contains the carb-oxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. Thec-subunit plays a profound role in the assembly of the complex. | iFEBS Journal Identification of a domain in the a-subunit of the oxaloacetate decarboxylase Na pump that accomplishes complex formation with the c-subunit Pius Dahinden Klaas M. Pos and Peter Dimroth Institute of Microbiology ETH Zurich ETH Honggerberg Zurich Switzerland Keywords association domain flexible linker peptide oxaloacetate decarboxylase protein-protein interaction sodium ion transport decarboxylase Correspondence P. Dimroth Institute of Microbiology ETH Zurich ETH Honggerberg Wolfgang-Pauli-Strasse 10 CH-8093 Zurich Switzerland E-mail dimroth@ Present address Institute of Physiology University of Zurich Winterthurerstrasse 190 CH-8057 Zurich Switzerland Received 18 October 2004 revised 1 December 2004 accepted 10 December 2004 doi The oxaloacetate decarboxylase Na pumps OAD-1 and OAD-2 of Vibrio cholerae are composed of a peripheral a-subunit associated with two integral membrane-bound subunits b and y . The a-subunit contains the carboxyltransferase domain in its N-terminal portion and the biotin-binding domain in its C-terminal portion. The y-subunit plays a profound role in the assembly of the complex. It interacts with the b-subunit through its N-terminal membrane-spanning region and with the a-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 a-2 and the C-terminal domain of y-2 termed y -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 . An internal stretch of 40 amino acids of a-2 was identified by deletion mutagenesis to be essential for the binding with y -2. This portion of the a-sub-unit 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 .