ON THE MODE OF INTEGRATION OF PLASTID-ENCODED COMPONENTS OF THE CYTOCHROME BF COMPLEX INTO THYLAKOID MEMBRANES

Four distinct integration/translocation routes into/across thylakoid m embranes have recently been deduced for nuclear-encoded polypeptides o f the photosynthetic membrane. Corresponding information for the plast id-encoded protein complement is lacking. We have investigated this as pect with in-organello assays employing chimeric constructs generated with codon-correct cassettes for genes of plastid-encoded thylakoid pr oteins, and appropriate transit peptides from six nuclear genes, repre senting three targeting classes, as a strategy. The three major plasti d-encoded components of the cytochrome b(6)f complex, namely pre-apocy tochrome f, (including apocytochrome f, and pre-apocytochrome f lackin g the C-terminal transmembrane segment). cytochrome b(6), and subunit IV, which differ in the number of their transmembrane segments, were s tudied. Import into chloroplasts could be observed in all instances bu t with relatively low efficiency. Thylakoid integration can occurr pos t-translationally, but only components with secretory/secretory pathwa y (SEC)-route-specific epitopes were correctly assembled with the cyto chrome complex, or competed with this process. Inhibitor studies were consistent with these findings. Imported cytochrome b(6) and subunit I V operated with uncleaved targeting signals for thylakoid integration. The corresponding determinant for cytochrome f is its signal peptide; its C-terminal hydrophobic segment did not, or did not appreciably. c ontribute to this process. The N-termini of cytochrome b(6) and subuni t IV appear to reside on the same (lumenal) side of the membrane, cons istent with the currently favored four-helix model for the cytochrome, but in disagreement with the topography proposed for both components. The impact of the findings for protein routing, including for applied approaches such as compartment-alien transformation, is discussed.