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Botanisches Institut, Universität Kiel, 24118 Kiel, Germany
The chloroplastic inner envelope protein of
110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of
IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the
mutual exchange of presequences with the precursor of
the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic
inner envelope is contained in an NH2-proximal part of
mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80-90 kD) are exposed
to the intermembrane space in situ. Successful sorting
and integration of IEP110 and the derived constructs
into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease
thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space.
A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and
mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be
recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport
pathway, which results not 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 reexport machinery for IEPs
in which soluble stromal components interact with a
membrane-localized translocation machinery.
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