Abstract
The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.
Highlights
The spinach triose phosphate/phosphate translocator and the 37·kDa protein are both integral components of the chloroplast inner envelope membrane
In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll alb binding protein and the CFoIl-subunit of the thylakoid ATP synthase, respectively
For the precursor proteins that are located to the interior of chloroplasts, it has been demonstrated that the information for targeting to the chloroplast and translocation across the envelope is present in N-terminal transit sequences
Summary
Vol 270, No 35, Issue of September 1, pp, 20808-20815, 1995 Printed in U.S.A. Sorting of Nuclear-encoded Chloroplast Membrane Proteins to the Envelope and the Thylakoid Membrane*. The spinach triose phosphate/phosphate translocator and the 37·kDa protein are both integral components of the chloroplast inner envelope membrane They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In organello import studies on two prominent components of this compartment, i.e. the triose phosphate/phosphate translocator (TPT) and the 37-kDa protein, had revealed that the post-translational import of these nuclear-encoded proteins involves, similar to the import of stromal or thylakoid proteins, binding of precursors to the outer envelope membrane, ATP-dependent translocation into the organelle, and proteolytic removal of the transit peptide (Fliigge et al, 1989; Dreses-Werringloer et al, 1991; Willey et al, 1991). We have produced a series of chimeric proteins in which the transit peptides and mature parts of proteins of the envelope and thylakoid membranes were reciprocally exchanged and compared the import and intraorganellar routing of these chimeras with that of the corresponding authentic proteins
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