MOLECULAR CHARACTERIZATION OF THE 50 AND 57-KDA SUBUNITS OF THE BOVINE VACUOLAR PROTON PUMP

The vacuolar type proton-translocating ATPase of clathrin-coated vesic les is composed of two large domains: an extramembranous catalytic sec tor and a transmembranous proton channel, In addition, two polypeptide s of 50 and 57 kDa have been found to copurify with the pump, These pr oteins, termed SFD (sub-fifty-eight-kDa dimer) activate ATPase activit y of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Chem B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem, 269, 285 09-25818). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex resp onsible for the assembly of clathrin-coated pits, and that AP50 is ess ential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592-31597), We demonstrate through th e use of anti-AP50 antibody, identical to that of the latter study, th at hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional. To determine the true molecular identity of SFD, both the 50- and 57-kDa polpeptides were directly sequenced. A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA librar y, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a re verse transcription-polymerase chain reaction-based strategy to isolat e a related, but distinct, transcript present in bovine brain mRNA. Th e nucleotide and predicted amino acid sequences of this isolate are id entical to SFD-21 except, that the isolate contains a 54-base pair ins ert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da. Both clones had 16% identity to VMA13 of Saccharom yces cerevisiae. No sequence homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epit ope common to the two proteins and to the unique 18-amimao acid insert of tile larger protein, The former reacted with both components of na tive SFD, whereas the latter reacted only with the 57-kDa component, W e term the 57- and 50-kDa polypeptides SFD alpha and SFD beta, respect ively.