title: Stable association of chloroplastic precursors with protein translocation complexes that contain proteins from both envelope membranes and a stromal Hsp100 molecular chaperone.
abstract: Cytoplasmically synthesized precursors interact with translocation components in both the outer and inner envelope membranes during transport into chloroplasts. Using co-immunoprecipitation techniques, with antibodies specific to known translocation components, we identified stable interactions between precursor proteins and their associated membrane translocation components in detergent-solubilized chloroplastic membrane fractions. Antibodies specific to the outer envelope translocation components OEP75 and OEP34, the inner envelope translocation component IEP110 and the stromal Hsp100, ClpC, specifically co-immunoprecipitated precursor proteins under limiting ATP conditions, a stage we have called docking. A portion of these same translocation components was co-immunoprecipitated as a complex, and could also be detected by co-sedimentation through a sucrose density gradient. ClpC was observed only in complexes with those precursors utilizing the general import apparatus, and its interaction with precursor-containing translocation complexes was destabilized by ATP. Finally, ClpC was co-immunoprecipitated with a portion of the translocation components of both outer and inner envelope membranes, even in the absence of added precursors. We discuss possible roles for stromal Hsp100 in protein import and mechanisms of precursor binding in chloroplasts.
authors.affiliation: MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing 48824, USA.
authors.forename: E
authors.initials: E
authors.lastname: Nielsen
authors.forename: M
authors.initials: M
authors.lastname: Akita
authors.forename: J
authors.initials: J
authors.lastname: Davila-Aponte
authors.forename: K
authors.initials: K
authors.lastname: Keegstra
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D002736:Chloroplasts / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006360:Heat-Shock Proteins / Q000378:metabolism*; D011495:Histone-Lysine N-Methyltransferase / Q000378:metabolism; D015151:Immunoblotting; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D018532:Pisum sativum / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism
chemical_list: D003902:Detergents; D006360:Heat-Shock Proteins; C102167:IEP110 protein, Pisum sativum; D008565:Membrane Proteins; D018832:Molecular Chaperones; C103933:OEP75 protein precursor, plant; D010940:Plant Proteins; D011498:Protein Precursors; D000255:Adenosine Triphosphate; C097344:ribulose-1,5-bisphosphate carboxylase-oxygenase large subunit epsilonN-methyltransferase; D011495:Histone-Lysine N-Methyltransferase