Import of barley photosystem I subunit N into the thylakoid lumen is mediated by a bipartite presequence lacking an intermediate processing site. Role of the delta pH in translocation across the thylakoid membrane.
Translocation across the thylakoid membrane of the recently identified photosystem I polypeptide, PSI-N, has been analyzed in pea (Pisum sativum) and barley (Hordeum vulgare). PSI-N from barley is synthesized in the cytosol with a bipartite presequence similar in structural terms to those of other cytosolically synthesized proteins routed to the thylakoid lumen. In vitro reconstitution assays demonstrate that translocation into thylakoids is absolutely dependent on the transthylakoidal delta pH, but that nucleotide triphosphates are not required; the translocation mechanism is thus similar in these respects to those utilized by the 23- and 16-kDa proteins of the oxygen-evolving complex. The translocation of PSI-N is unique in that the presequence of PSI-N does not contain an intermediate cleavage site for the stromal processing peptidase; important experiments using intact chloroplasts depleted of a delta pH by nigericin treatment demonstrate the accumulation of the full precursor protein in the stroma. Translocation across the thylakoid membrane can take place in the absence of stromal factors, although the presence of stromal extracts leads to a consistent but slight enhancement of translocation efficiency. We also show that efficient translocation of the 33-kDa protein of the oxygen-evolving complex can take place in the complete absence of a delta pH, in apparent contradiction with earlier findings; the translocation of this protein is thus similar in several respects to that of plastocyanin. The data indicate the operation of two very different types of translocation mechanism, with PSI-N exhibiting additional separate characteristics.
Nielsen
V S
VS
Department of Plant Biology, Royal Veterinary and Agricultural University, Frederiksberg C, Copenhagen, Denmark.
Mant
A
A
Knoetzel
J
J
Møller
B L
BL
Robinson
C
C
D002736:Chloroplasts / Q000187:drug effects / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D007887:Fabaceae / Q000378:metabolism*; D001467:Hordeum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism; D046911:Macromolecular Substances; D009550:Nigericin / Q000494:pharmacology; D015388:Organelles / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000096:biosynthesis*; D045331:Photosystem I Protein Complex; D010946:Plants, Medicinal; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D046911:Macromolecular Substances; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011498:Protein Precursors; D010450:Endopeptidases; D009550:Nigericin