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.
Departamento de Ciencias Biológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
J
J
Ottado
A K
AK
Arakaki
N B
NB
Calcaterra
E A
EA
Ceccarelli
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