INTERACTION OF THE PROTEIN IMPORT AND FOLDING MACHINERIES IN THE CHLOROPLAST

We report the molecular cloning of import intermediate associated prot ein (IAP) 100, a 100-kDa protein of the chloroplast protein import mac hinery of peas, IAP100 contains two potential alpha-helical transmembr ane segments and also behaves like an integral membrane protein, It wa s localized to the inner chloroplast envelope membrane, Immunoprecipit ation experiments using monospecific anti-IAP100 antibodies and a noni onic detergent-generated chloroplast lysate gave the following results , (i) The four integral membrane proteins of the outer chloroplast imp ort machinery were not coprecipitated with IAP100 indicating that the inner and outer membrane import machineries are not coupled in isolate d chloroplasts. (ii) the major protein that coprecipitated with IAP100 was identified as stromal chaperonin 60 (cpn60); the association of I AP100 and cpn60 was specific and was abolished when immunoprecipitatio n vias carried out in the presence of ATP, (iii) In a lysate from chlo roplasts 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 th e imported mature form (S) of precursor, Relative to the time course o f import, coprecipitation of S first increased and then decreased, con sistent with a transient association of the newly imported S with the chaperonin bound to IAP100. These data suggest that IAP100 serves in r ecruiting chaperonin for folding of newly imported proteins.