In vitro integration of the ternary complex, POR-Pchlide-NADPH, into pea thylakoids

Abstract

The nucleus-encoded NADPH: protochlorophyllide oxidoreductase (POR), catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) in higher plants. This is the first step in the biosynthetic pathway of chlorophyll that requires light. POR is mainly found in the prolamellar bodies (PLBs) in dark-grown plants but also at lower levels in greening plants. In barley and Arabidopsis thaliana two different PORs, POR-A and POR-B, have been found. POR-A has been suggested to be dependent upon the substrate, Pchlide, for translocation into the plastid [1]. After import, POR is assembled on the stromal side of the plastid inner membranes in accordance with the lack of transfer sequences for lumen translocation or membrane spanning regions. The catalytically active form is a ternary complex consisting of POR, Pchlide and NADPH. By analogy with the import reaction it could be speculated that Pchlide could play an active role in the interaction with the plastid inner membranes, e.g. is the integration facilitated by a pre-assembly of a ternary complex before the membrane integration? To address this question, we have used an in vitro integration assay to examine the requirement for ternary complex formation as a pre-requisite for membrane assembly of mature POR. We have also examined how mutations within POR affecting NADPH and Pchlide binding capacity (and thus ternary complex formation) affect integration. Our data suggest that it can not be excluded that a formation of a ternary complex in the stroma precede the integration reaction. However, the equally efficient integration of free POR, in the absence of exogenous NADPH and Pchlide, suggests that alternative membrane targeting pathways may exist.