Interaction of the protein import and folding machineries of the chloroplast.
We report the molecular cloning of import intermediate associated protein (IAP) 100, a 100-kDa protein of the chloroplast protein import machinery of peas. IAP100 contains two potential alpha-helical transmembrane segments and also behaves like an integral membrane protein. It was localized to the inner chloroplast envelope membrane. Immunoprecipitation experiments using monospecific anti-IAP100 antibodies and a nonionic detergent-generated chloroplast lysate gave the following results. (i) The four integral membrane proteins of the outer chloroplast import machinery were not coprecipitated with IAP100 indicating that the inner and outer membrane import machineries are not coupled in isolated chloroplasts. (ii) the major protein that coprecipitated with IAP100 was identified as stromal chaperonin 60 (cpn60); the association of IAP100 and cpn60 was specific and was abolished when immunoprecipitation was carried out in the presence of ATP. (iii) In a lysate from chloroplasts that had been preincubated for various lengths of time in an import reaction with radiolabeled precursor (pS) of the small subunit of Rubisco, we detected coimmunoprecipitation of IAP100, cpn60, and the imported mature form (S) of precursor. Relative to the time course of import, coprecipitation of S first increased and then decreased, consistent with a transient association of the newly imported S with the chaperonin bound to IAP100. These data suggest that IAP100 serves in recruiting chaperonin for folding of newly imported proteins.
Kessler
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F
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.
Blobel
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G
D000595:Amino Acid Sequence; D001345:Autoradiography; D001483:Base Sequence; D018834:Chaperonin 60 / Q000302:isolation & purification / Q000378:metabolism; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D018532:Pisum sativum; D011485:Protein Binding; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013462:Sulfur Radioisotopes
D018834:Chaperonin 60; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; C101484:import intermediate associated protein 100