A novel plasma membrane-bound thioredoxin from soybean.
Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3 beta-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.
Shi
J
J
Plant Biology Division, Samuel Roberts Noble Foundation, Ardmore, OK 73402, USA.
Bhattacharyya
M K
MK
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D018744:DNA, Plant / Q000235:genetics; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000378:metabolism; D000939:Epitopes; D007328:Insulin / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010455:Peptides / Q000235:genetics; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D019163:Reducing Agents / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000737:chemistry / Q000235:genetics*; D013879:Thioredoxins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics
D018076:DNA, Complementary; D018744:DNA, Plant; D004220:Disulfides; D000939:Epitopes; D007328:Insulin; D008565:Membrane Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D013879:Thioredoxins; C085706:FLAG peptide; D004229:Dithiothreitol