Plant Molecular Biology 32: 653~562, 1996.
(~) 1996KluwerAcademic Publishers. Printedin Belgium.
653
A novel plasma membrane-bound thioredoxin from soybean
J i n r u i Shi a n d M a d a n K. B h a t t a c h a r y y a *
Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, OK 73402, USA
(*author for correspondence)
Received 13 February 1996;acceptedin revisedform20 June 1996
Key words: Glycine max (L.) Merr., epitope-tagging, immunoscreening, developmental regulation, transmembrane
domain
Abstract
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 1,3/3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane
location of TRX 1. 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 TRXI
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.
Introduction
Thioredoxins are small proteins of ca. 12 kDa which
catalyze thiol-disulfide reduction-oxidation reactions
through a disulfide group interchange (between SS and 2SH). Amino acid and nucleotide sequences
have been obtained for thioredoxins from different
prokaryotic and eukaryotic species. A disulfide active
site, -Cys-Gly-Pro-Cys-, is highly conserved in most
thioredoxins [ 14].
Thioredoxins participate in many diverse biochemical processes, such as protein folding [27], protection of cellular components against oxidative damage [5, 6, 19], activation of cytosolic glucocorticoid
receptor [ 12], promoting the growth of lymphoid cells
through activation of the phosphoinositide signaling
pathway [3], regulation of transcription factors NF-~B
and AP-1 [31], assembly of filamentous phage [30],
T7 DNA polymerase-catalyzed DNA biosynthesis [25]
and development of nitrogen-fixing symbioses [21 ]. In
higher plants, three groups of thioredoxins have been
identified. The f-type and m-type plant thioredoxins
occur in chloroplasts, serve as electron donors, and are
involved in light regulation of photosynthetic enzymes
[ 18, 23]. The h-type plant thioredoxins have multiple
subcellular locations, including cytosol, endoplasmic
reticulum (ER), and mitochondria [24]. Recently, an
h-type thioredoxin was identified in rice phloem sap.
This protein is one of the major phloem sap proteins
[ 17]. The h-type plant thioredoxins are closely related
to mammalian thioredoxins in terms of amino acid
sequence similarity [24]. In many mammalian species, thioredoxin appears to be encoded by a single
gene. Plants, however, contain divergent cytoplasmic