2 DISTINCT MECHANISMS FOR THE TRANSLOCATION OF PROTEINS ACROSS THE THYLAKOID MEMBRANE, ONE REQUIRING THE PRESENCE OF A STROMAL PROTEIN FACTOR AND NUCLEOTIDE TRIPHOSPHATES

Cytosolically synthesized thylakoid lumen proteins are normally transported across the thylakoid membrane of chloroplasts as intermediate forms following cleavage Of the full PreCursor polypeptides by a stromal peptidase. We have examined the mechanisms by which two lumenal proteins, the 33- and 23-kDa components of the oxygen-evolving complex, are translocated across the thylakoid membrane using an in vitro import assay and synthetic engineered intermediate forms of the two proteins as substrates. Efficient transport of the 23-kDa protein is observed in both the presence and absence of stromal extracts, but efficient import of the 33-kDa protein into thylakoids is completely dependent on the presence of stroma. The required stromal factor is heat-sensitive, macromolecular, and trypsin-sensitive. These findings thus represent the first evidence for the active participation of one or more stromal proteins in thylakoidal protein transport. We also show that import into thylakoids of the 33-kDa protein, but not of the 23-kDa protein, is absolutely dependent on the presence of nucleotide triphosphates. The data therefore indicate that at least two distinct mechanisms exist for the translocation of Iumenal proteins across the thylakoid membrane