Topology mapping of the vacuolar Vcx1p Ca2+/H+ exchanger from Saccharomyces cerevisiae.
Saccharomyces cerevisiae uses vacuolar storage to dynamically control the cytoplasmic calcium concentration. Vcx1p, a Ca(2+)/H(+) antiporter and a member of the CAX (Ca(2+)/anion exchanger) family of exchangers, is one of the proteins that sequesters calcium into the vacuole. Although the biological importance of Vcx1p is clear, the molecular mechanism by which Vcx1p and its family members mediate Ca(2+)/H(+) exchange activity remains poorly understood. To provide a basic structural framework for understanding functional studies of the CAX proteins, we have mapped Vcx1p's topology using three biochemical assays: C-terminal reporter localization, glycosylation mapping and proteolysis. We have found that the protein has an odd number of TM (transmembrane) domains and that its termini are located on opposite sides of the membrane, with the N-terminus in the cytoplasm. Our results indicate that loops 1, 3, 7 and 9 are luminal, while loops 6 and 8 are cytosolic. Our experimentally-based topology model for Vcx1p is in agreement with models derived from topology algorithms and with biochemical data reported by other groups. In addition, our studies suggest that the calcium domain, a nine-residue domain found to be critical for function in CAX proteins from plants, is not essential to Vcx1p activity.
Segarra
Verónica A
VA
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT 06517, USA.
Thomas
Lise
L
D017920:Antiporters / Q000737:chemistry* / Q000502:physiology; D003593:Cytoplasm / Q000201:enzymology; D006031:Glycosylation; D006868:Hydrolysis; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000502:physiology; D019831:Sodium-Calcium Exchanger / Q000737:chemistry* / Q000502:physiology; D014617:Vacuoles / Q000201:enzymology* / Q000235:genetics*
D017920:Antiporters; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D019831:Sodium-Calcium Exchanger; C099946:VCX1 protein, S cerevisiae