DIFFERENTIAL EXPOSURE OF SURFACE EPITOPES IN THE BETA-STRAND REGION OF LOOP1 OF THE YEAST H-ATPASE DURING CATALYSIS()

The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural ch anges in the LOOP1 domain, a catalytically important cytoplasmic loop segment Linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis. A polyclonal rabbit antibod y was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOO P1-specific fraction that could be used in competition enzyme-linked i mmunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E-1 conformation stabilized with either adenos ine 5'-(beta,gamma-imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme. However, when the enzyme was stabilized in an E-w-state with ATP plus vanadate, appr oximately 40% of the surface epitopes on LOOP1 became accessible to an tibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked im munosorbent assays utilizing fusion proteins consisting of LOOP1 subdo mains demonstrated that all of the available epitopes were contained i n the beta-strand region (Glu-195-Val-267) of LOOP1, The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TOES sequence. Our re sults suggest that during catalysis either the beta-strand region of L OOP1 or an interacting domain undergoes substantial structural rearran gement that facilitates epitope exposure.