Association of the NADPH:protochlorophyllide oxidoreductase (POR) with isolated etioplast inner membranes from wheat.
Membrane association of NADPH:protochlorophyllide oxidoreductase (POR, EC: 1.6.99.1) with isolated prolamellar bodies (PLBs) and prothylakoids (PTs) from wheat etioplasts was investigated. In vitro-expressed radiolabelled POR, with or without transit peptide, was used to characterize membrane association conditions. Proper association of POR with PLBs and PTs did not require the presequence, whereas NADPH and hydrolysable ATP were vital for the process. After treating the membranes with thermolysin, sodium hydroxide or carbonate, a firm attachment of the POR protein to the membrane was found. Although the PLBs and PTs differ significantly in their relative amount of POR in vivo, no major differences in POR association capacity could be observed between the two membrane systems when exogenous NADPH was added. Experiments run with only an endogenous NADPH source almost abolished association of POR with both PLBs and PTs. In addition, POR protein carrying a mutation in the putative nucleotide-binding site (ALA06) was unable to bind to the inner membranes in the presence of NADPH, which further demonstrates that the co-factor is essential for proper membrane association. POR protein carrying a mutation in the substrate-binding site (ALA24) showed less binding to the membranes as compared to the wild type. The results presented here introduce studies of a novel area of protein-membrane interaction, namely the association of proteins with a paracrystalline membrane structure, the PLB.
Engdahl
S
S
Department of Plant Physiology, Göteborg University, Box 461, SE-405 30 Göteborg, Sweden.
Aronsson
H
H
Sundqvist
C
C
Timko
M P
MP
Dahlin
C
C
D000255:Adenosine Triphosphate / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects / Q000201:enzymology*; D009154:Mutation; D009249:NADP / Q000494:pharmacology; D015388:Organelles / Q000187:drug effects / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D018087:Plastids; D013379:Substrate Specificity; D014908:Triticum / Q000235:genetics / Q000378:metabolism*
D009249:NADP; D000255:Adenosine Triphosphate; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase