A NUCLEAR-CODED CHLOROPLASTIC INNER ENVELOPE MEMBRANE-PROTEIN USES A SOLUBLE SORTING INTERMEDIATE UPON IMPORT INTO THE ORGANELLE

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins we re constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chlor oplasts, as shown by the mutual exchange of presequences with the prec ursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (p SSU). Sorting information to the chloroplastic inner envelope is conta ined in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal p ortions of this protein (80-90 kD) are exposed to the intermembrane sp ace in situ. Successful sorting and integration of IEP110 and the deri ved constructs into the inner envelope are demonstrated by the inacces sability of processed mature protein to the protease thermolysin but a ccessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequen ce of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpS SU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110N-mSSU. The soluble 110N-mSSU then enters a reexport pathway, which results n ot only in the insertion of 110N-mSSU into the inner envelope membrane , but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein r eexport machinery for IEPs in which soluble stromal components interac t with a membrane-localized translocation machinery.