Biochemically characterizing the subcellular localization of peroxisomal proteins by fractionation, protease protection, and carbonate extraction.
Traditional biochemical approaches, as well as the complementary methods of living cell fluorescence microscopy and immunofluorescence microscopy, can serve to characterize the subcellular localization of proteins and organelles. This chapter describes methods for isolation of crude organelle fractions from methanol- or oleate-grown Pichia pastoris, followed by protease protection and carbonate extraction assays to dissect the subcellular localization of peroxisomal matrix and membrane proteins. These biochemical tools can be used to analyze the targeting efficiency of proteins to the peroxisome membrane and matrix, as well as the topology of membrane proteins.
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, 92093-0322, USA.
Danielle
D
Hagstrom
Changle
C
Ma
D002254:Carbonates / Q000737:chemistry; D010450:Endopeptidases; D005656:Fungal Proteins / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D007425:Intracellular Membranes; D008967:Molecular Biology / Q000379:methods*; D010447:Peptide Hydrolases / Q000737:chemistry; D020675:Peroxisomes / Q000378:metabolism; D010843:Pichia / Q000166:cytology* / Q000378:metabolism
D002254:Carbonates; D005656:Fungal Proteins; D010450:Endopeptidases; D010447:Peptide Hydrolases