title: Protein trafficking to the complex chloroplasts of Euglena.
abstract: Proteins are delivered to Euglena chloroplasts using the secretory pathway. We describe analytical methods to study the intracellular trafficking of Euglena chloroplast proteins and a method to isolate preparative amounts of intact import competent chloroplasts for biochemical studies. Cells are pulse labeled with 35S-sulfate and chased with unlabeled sulfate allowing the trafficking and posttranslational processing of the labeled protein to be followed. Sucrose gradients are used to separate a 35S-labeled cell lysate into cytoplasmic, endoplasmic reticuum (ER), Golgi apparatus, chloroplast and mitochondrial fractions. Immunoprecipitation of each gradient fraction allows identification of the intracellular compartment containing a specific 35S-labeled protein at different times after synthesis delineating the trafficking pathway. Because sucrose gradients cannot be used to isolate preparative amounts of highly purified chloroplasts for biochemical characterization, a preparative high-yield procedure using Percoll gradients to isolate highly purified import competent chloroplasts is also presented.
authors.affiliation: Institute of Cell Biology, Comenius University, Bratislava, Slovakia.
authors.forename: Rostislav
authors.initials: R
authors.lastname: Vacula
authors.forename: Silvia
authors.initials: S
authors.lastname: Sláviková
authors.forename: Steven D
authors.initials: SD
authors.lastname: Schwartzbach
mesh_terms: D000818:Animals; D002458:Cell Fractionation; D002736:Chloroplasts / Q000378:metabolism*; D005055:Euglena / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D021381:Protein Transport; D013347:Subcellular Fractions
chemical_list: D010940:Plant Proteins