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J Biol Chem, Vol. 273, Issue 29, 18282-18287, July 17, 1998

Differential Exposure of Surface Epitopes in the -Strand Region of LOOP1 of the Yeast H⁺-ATPase during Catalysis

From the Public Health Research Institute, New York, New York 10016

The plasma membrane H⁺-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural changes 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 antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E₁ conformation stabilized with either adenosine 5'-(,-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 E₂-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the -strand region (Glu-195Val-267) of LOOP1. The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence. Our results suggest that during catalysis either the -strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.

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