Protein trafficking to the complex chloroplasts of Euglena.
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.
Vacula
Rostislav
R
Institute of Cell Biology, Comenius University, Bratislava, Slovakia.
Sláviková
Silvia
S
Schwartzbach
Steven D
SD
D000818:Animals; D002458:Cell Fractionation; D002736:Chloroplasts / Q000378:metabolism*; D005055:Euglena / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D021381:Protein Transport; D013347:Subcellular Fractions
D010940:Plant Proteins