ENZYMATIC PRODUCT FORMATION IMPAIRS BOTH THE CHLOROPLAST RECEPTOR-BINDING FUNCTION AS WELL AS TRANSLOCATION COMPETENCE OF THE NADPH - PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, A NUCLEAR-ENCODED PLASTID PRECURSOR PROTEIN
S. Reinbothe et al., ENZYMATIC PRODUCT FORMATION IMPAIRS BOTH THE CHLOROPLAST RECEPTOR-BINDING FUNCTION AS WELL AS TRANSLOCATION COMPETENCE OF THE NADPH - PROTOCHLOROPHYLLIDE OXIDOREDUCTASE, A NUCLEAR-ENCODED PLASTID PRECURSOR PROTEIN, The Journal of cell biology, 129(2), 1995, pp. 299-308
The key enzyme of chlorophyll biosynthesis in higher plants, the light
-dependent NADPH:protochlorophyllide oxidoreductase (FOR, EC 1.6.99.1)
, is a nuclear-encoded plastid protein. Its posttranslational transpor
t into plastids of barley depends on the intraplastidic availability o
f one of its substrates, protochlorophyllide (PChlide). The precursor
of FOR (pPOR), synthesized from a corresponding full-length barley cDN
A clone by coupling in vitro transcription and translation, is enzymat
ically active and converts PChlide to chlorophyllide (Chlide) in a lig
ht- and NADPH-dependent manner. Chlorophyllide formed catalytically re
mains tightly but noncovalently bound to the precursor protein and sta
bilizes a transport-incompetent conformation of pPOR. As shown by in v
itro processing experiments, the chloroplast transit peptide in the Ch
lide-pPOR complex appears to be masked and thus is unable to physicall
y interact with the outer plastid envelope membrane. In contrast, the
chloroplast transit peptide in the naked pPOR (without its substrates
and its product attached to it) and in the pPOR-substrate complexes, s
uch as pPOR-PChlide or pPOR-PChlide-NADPH, seems to react independentl
y of the mature region of the polypeptide, and thus is able to bind to
the plastid envelope. When envelope-bound pPOR-PChlide-NADPH complexe
s were exposed to light during a short preincubation, the enzymaticall
y produced Chlide slowed down the actual translocation step, giving ri
se to the sequential appearance of two partially processed translocati
on intermediates. However, ongoing translocation induced by feeding th
e chloroplasts delta-aminolevulinic acid, a precursor of PChlide, was
able to override these two early blocks in translocation, suggesting t
hat the plastid import machinery has a substantial capacity to denatur
e a tightly folded, envelope-bound precursor protein. Together, our re
sults show that pPOR with Chlide attached to it is impaired both in th
e ATP-dependent step of binding to a receptor protein component of the
outer chloroplast envelope membrane, as well as in the PChlide-depend
ent step of precursor translocation.