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 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.