Expression, assembly and secretion of a fully active plant ferredoxin-NADP+ reductase by Saccharomyces cerevisiae.
The flavoprotein ferredoxin-NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin. Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library [Newman, B. J. & Gray, J. C. (1988) Plant Mol. Biol. 10, 511-520]. Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin-NADP+ reductase to the chloroplast. These sequences were introduced into a yeast shuttle vector in frame with the mating factor alpha 1 secretion-signal coding region under the control of its natural mating factor alpha 1 promoter. Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin-NADP+ reductase. In both cases, a 35-kDa polypeptide was the major product. N-terminal sequencing of the secreted proteins indicates processing at position -1 with respect to the N-terminus of the pea mature enzyme. Yeast cells transformed with plasmid encoding the ferredoxin-NADP+ reductase precursor secrete four-times more ferredoxin-NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme. Ferredoxin-NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin-NADP+ reductase. The overall results indicate that pea ferredoxin-NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion.
Ottado
J
J
Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
Arakaki
A K
AK
Calcaterra
N B
NB
Ceccarelli
E A
EA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology*; D010957:Plasmids; D017510:Protein Folding; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D005287:Ferredoxin-NADP Reductase