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 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'-(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 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 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 TGES sequence. Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.
Seto-Young
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Public Health Research Institute, New York, New York 10016, USA.
Bandell
M
M
Hall
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M
Perlin
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DS
D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D004797:Enzyme-Linked Immunosorbent Assay; D018604:Epitope Mapping; D007527:Isoenzymes / Q000378:metabolism*; D058619:Maltose-Binding Proteins; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins
D002352:Carrier Proteins; D007527:Isoenzymes; D058619:Maltose-Binding Proteins; C434364:PMA2 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000266:Adenylyl Imidodiphosphate; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases