IDENTIFICATION OF A 14-KDA SUBUNIT ASSOCIATED WITH THE CATALYTIC SECTOR OF CLATHRIN-COATED VESICLE H-ATPASE()

The clathrin-coated vesicle H+-ATPase is composed of a peripheral cata lytic sector (V-C) and an integral membrane proton channel (V-B), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and seque nced cDNA clones from bovine and rat brain cDNA libraries. A full-leng th clone from rat brain encodes a 119-amino acid polypeptide with a pr edicted molecular mass of 13,370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Sou thern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced p rotein sequence, was affinity-purified and shown to react with a 14-kD a polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated V-C. When stripped clathrin -coated vacuolars and purified chromaffin granule membranes were treat ed with KI in the presence of ATP, the 14 kDa subunit was released fro m both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa compo nent of the coated vacuolar proton pump confirmed its identity as a su bunit F homologue.