E. Zak et al., ON THE MODE OF INTEGRATION OF PLASTID-ENCODED COMPONENTS OF THE CYTOCHROME BF COMPLEX INTO THYLAKOID MEMBRANES, Planta, 201(3), 1997, pp. 334-341
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.