Localization of a protein A-tagged Kex2 protein to the vacuole of Saccharomyces cerevisiae allows rapid purification of vacuolar membranes.
We have previously reported an immunoisolation procedure which allows purification of Kex2p-containing Golgi membranes from lysed yeast cells. In order to evaluate the use of tagging procedures in organelle isolation we set out to isolate the same Golgi membrane fraction using a version of the Kex2 protease that had been affinity-tagged at its C-terminus. This protein is found to be localized in the vacuole, providing the basis of a method for the affinity-purification of vacuolar membranes.
Bryant
N J
NJ
Department of Biochemistry, University of Edinburgh, U.K.
Boyd
A
A
D001483:Base Sequence; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D007425:Intracellular Membranes / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D043484:Proprotein Convertases; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D012685:Sepharose / Q000031:analogs & derivatives; D013205:Staphylococcal Protein A; D013347:Subcellular Fractions / Q000201:enzymology; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*; D014617:Vacuoles / Q000201:enzymology*
D017931:DNA Primers; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D013205:Staphylococcal Protein A; C036210:immunoglobulin G-sepharose; D012685:Sepharose; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae