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Plant Physiol. (1998) 118: 987-996
A Novel Nuclear Member of the Thioredoxin Superfamily
Beth J. Laughner,
Paul C. Sehnke, and
Robert J. Ferl*
Plant Molecular and Cellular Biology Program, Department of
Horticultural Sciences, University of Florida, Gainesville, Florida
32611
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ABSTRACT |
We describe the isolation and
characterization of a cDNA encoding maize (Zea mays L.)
nucleoredoxin (NRX), a novel nuclear protein that is a member of the
thioredoxin (TRX) superfamily. NRX is composed of three TRX-like
modules arranged as direct repeats of the classic TRX domain. The first
and third modules contain the amino acid sequence WCPPC, which
indicates the potential for TRX oxidoreductase activity, and insulin
reduction assays indicate that at least the third module possesses TRX
enzymatic activity. The carboxy terminus of NRX is a non-TRX
module that possesses C residues in the proper sequence context to form
a Zn finger. Immunolocalization preferentially to the nucleus within
developing maize kernels suggests a potential for directed alteration
of the reduction state of transcription factors as part of the events and pathways that regulate gene transcription.
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INTRODUCTION |
The concept of redox regulation is becoming increasingly important
at diverse levels of cellular function. Under oxidative stress, cells
exposed to reactive oxygen species without appropriate buffering
capacity could die. Moderation of the redox state is an important
function in intracellular signaling (Nakamura et al., 1997 ).
Furthermore, the binding of several transcription factors, such as NFB
66 and AP-1, has been shown to be markedly influenced by their
respective redox states (Schenk et al., 1994; Hirota et al., 1997).
TRXs are typically small, approximately 12-kD proteins that participate
in cellular redox reactions. They are widely distributed, being present
in some form in all organisms, from bacteria and yeast to animals and
plants. Plants have multiple family members, including plant-specific
forms that are found in the chloroplast (the TRX-f and TRX-m forms), as
well as TRX-h, which is more animal-like in sequence and is found in
the cytosol, ER, and mitochondria, as determined by
cellular-fractionation studies (Marcus et al., 1991; Buchanan et al.,
1994). Although multiple family members were thought to be unique to
plants, recent reports document larger TRXs such as an 18-kD mammalian
TRX and a novel 15.5-kD Escherichia coli TRX
(Miranda-Vizuete et al., 1997; Spyrou et al., 1997).
The redox activities of TRXs are due to an active site that contains
vicinal C residues that can exist in a reduced or mutually oxidized,
disulfide bridge state. An active-site motif of WCGPC is conserved
among the classic TRXs of E. coli and many other species.
However, other active-site sequences exist, including a WCPPC motif
that has been identified in TRX and TRX-like sequences within diverse
organisms such as Arabidopsis, Caenorhabditis
elegans, and Mus musculus (Rivera-Madrid
et al., 1995; Kurooka et al., 1997). The classic TRX has its active
site protruding from a highly organized, globular structure composed of
five strands of  -sheets enclosed by four  -helices (Holmgren,
1985). Specific conserved residues suggest a conserved tertiary
structure, even though the amino acid sequence among TRXs in general
can vary from 26% to 67% sequence identity compared with E. coli TRX (Eklund et al., 1991).
TRX-like domains have been identified in many eukaryotic proteins
through sequence similarity. A gene from a self-incompatibility locus
in the grass Phalaris coerulescens has a functional TRX domain (Li et al., 1995). PDIs contain multiple TRX domains and are
responsible for multiple disulfide rearrangements in protein folding,
an activity not overtly present in single-module TRXs (Freedman et al.,
1994). Human PDI, for example, has two TRX modules that can be
identified by sequence homology. A third module of PDI lacks vicinal C
residues but retains a TRX fold, as determined by NMR analysis (Kemmink
et al., 1997). PDIs usually have the active site of WCGHC and typically
exhibit ER-targeting and -retention signals. A mouse
TRX-domain-containing protein found in the nucleus has a TRX domain
with the WCPPC active-site motif (Kurooka et al., 1997) and vestiges of
a second, inactive TRX-like domain. Thus, it appears that a major
diversifying mechanism among TRX-like molecules is to link active and
inactive TRX domains together, perhaps thereby increasing functional
diversity or specificity.
Classic functional roles associated with TRXs include thiol redox
regulation of enzymes. Chloroplast TRX members utilize Fd and Fd
reductase in light-mediated reactions that target enzymes of the
reductive pentose phosphate or Calvin cycle (Buchanan et al., 1994).
Animal TRX, E. coli TRX, and plant TRX-h utilize NADPH with
TRX reductase in redox regulation. Specific roles for TRX-h in plants
include the reduction of purothionin, which is a small, disulfide-rich
protein in wheat grains, and early signaling in seed germination
(Buchanan et al., 1994). Recent reports continue to elucidate the role
of TRX in the regulation of transcription factors through changes in
the state of critical C residues located within their DNA-binding
domains. These changes may be effected directly as in the case of
NF B or indirectly in AP-1 through Ref-1 (Hayashi et al., 1993;
Hirota et al., 1997). Although TRXs lack a recognized
nuclear-localization signal, they can be translocated from the
cytoplasm to the nucleus in response to stress (Hirota et al., 1997;
Nakamura et al., 1997).
We report here the isolation from maize of a cDNA encoding a novel,
multiple TRX-domain protein that we call NRX because it is highly
localized to the nucleus, is composed of multiple TRX-domain modules,
and in its recombinant form possesses TRX enzymatic activity.
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MATERIALS AND METHODS |
Screening for Factors That Influence DNA Binding
A partially purified preparation of the ARF (Anaerobic
Response Factor)-B2 DNA-binding activity (Ferl,
1990) was used for the production of a panel of hybridomas in
anticipation of recovering antibodies that influence DNA-binding
activities. Several hundred hybridoma cell supernatants were screened
by assaying the effect of the supernatant on bandshifts involving
ARF-B2 activity. Two supernatants that reduced DNA-binding activity
were subsequently used to screen a  -gt11 maize (Zea mays
L.) suspension-cell library (Clontech, Palo Alto, CA) as previously
described (Lu et al., 1992). One positive clone was purified to
homogeneity and its phage eluate was subjected to a PCR reaction with
-gt11 primers to recover the insert. A product approximately 860 bp
in length was restricted with EcoRI and cloned into
pCR2 18 to produce plasmid Z863 (Fig. 1).
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| Figure 1.
cDNA clones used for the contig assembly of maize
NRX. A, Contig assembled by merging clones. The first clone, Z863, was
identified by immunoscreening a -gt11 custom-made maize
suspension-cell library. This fragment was cloned into pUC 18, and the
insert was used to screen for longer clones. The clone C1300 in the
vector pUC 18 provided the carboxyl end of the cDNA. The ZmD1 clone was
cloned into pCR2 and represents the longest cDNA. The clone referred to
as D5 was obtained through a modified 5 -rapid amplification of cDNA
ends technique and was cloned into pCRBLUNT. The clone designated Z10
was generated through the use of engineered NdeI and
BamHI sites. The NdeI site coincides with
the first inframe ATG codon, and the BamHI site lies
just downstream of the stop codon.
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cDNA Clones
Since northern analysis suggested that Z863 was a partial cDNA
clone, the EcoRI insert from Z863 was random-prime
labeled (Boehringer Mannheim) as a probe for further screening. Several -gt11 clones were purified to homogeneity and their respective phage
eluates were used to generate PCR products for cloning into pCR2 and
pCRBLUNT (Invitrogen, Carlsbad, CA). This screen produced cDNA plasmids
ZmD1 and C1300 (Fig. 1).
A modification of a 5-rapid amplification of cDNA ends protocol (Jain
et al., 1992) was used to clone the 5 end of the cDNA clone. The
following antisense nested primers were used: RF 271 5
CCGAAGCAAAGACGACCT 3 (nucleotide positions 362-345) and RF 272 5
GAAAAGGGGACAGCCAAC 3 (nucleotide positions 429-412). The first strand
was prepared from total RNA isolated from the maize suspension culture.
PCR amplification with the 3 most of the nested primers (RF 272) was
performed with 1 cycle at 95° for 80 s, 45° for 5 min, and
72° for 2 min followed by 3 cycles at 95° for 40 s, 48° for
1 min, and 72° for 2 min. This first round was filtered through an
Ultrafree MC-regenerated cellulose membrane with a 30,000 nominal
Mr limit (Millipore) cutoff to remove
primers and dNTPs. The PCR amplification with the 5-most primer (RF
271) was performed with the following specifications: 95° for 2 min and 30 cycles at 95° for 1 min, 58° for 2 min, and 72° for 2 min. It is noteworthy that only Vent polymerase (New England Biolabs) amplified a product that was subcloned into pCR2 to yield
plasmid D5 (Fig. 1).
A composite cDNA clone Z10 was generated by merging the products of the
5 extension protocol (D5) and the longest clone (ZmD1) by a
modification of strand-overlap extension (Kim et al., 1996). First, D5
was used as the template to generate the amino end product of the
clone. The 5 sense oligo (primer 1) included an engineered NdeI and the sequence from nucleotide positions 119 to 136 of NRX, whereas the antisense oligo (primer 2) annealed near the 3 end
of D5, at nucleotide positions 344 to 325. Second, the template ZmD1
was used with a sense oligo (primer 3) at nucleotide positions 241 to
269 and an antisense oligo (primer 4) just downstream of the stop codon
near nucleotide positions 1876 to 1858 and included an engineered
BamHI site to generate the carboxy end of the clone. The
overlap area was approximately 100 bp and facilitated amplification of
a composite clone of 1757 bp when both products were subjected to a
second round of PCR using the 5-most sense oligo (primer 1) and the
3-most antisense oligo (primer 4). The composite PCR product was
immediately cloned into pCRBLUNT. This clone, representing a putative
full-length clone for expression studies, was designated Z10 (Fig. 1).
Sequence Analysis
Automated dideoxy chain termination on both strands of these
plasmids using Taq polymerase was performed on a DNA
sequencer (model ABI 373, Applied Biosystems). Computer analyses on DNA sequences were performed with MacVector utilities (IBI, Eastman Kodak)
and Geneworks (IntelliGenetics, Oxford Molecular Group, Oxford, UK).
The final composite sequence was generated by the merging sequences
from D5, the longest clone, ZmD1, and a shorter clone, C1300, that
contained a poly(A+) tail (Fig. 1). Homology
searches were done using BLAST software (Altschul et al., 1997).
Expression Studies in E. coli
For expression of the carboxy-terminal portion of NRX ( NRX),
the EcoRI fragment of Z863 was subcloned into pETH 3A (McCarty et al., 1991) and, after sequence confirmation, the construct was transformed into BL21DE3 for protein expression. This construct was
referred to as Z10-14 and includes the carboxy-terminal portion of the
NRX protein underlined in Figure 2. The NRX coding sequence starts
22 amino acids from the NdeI site in the pETH vector, so the
resulting NRX is a fusion protein that includes the following deduced
amino acids from the vector as an amino-terminal leader: MASMTGGQQMGRSSFPGSSNSG. This fusion construct was later moved as a
NdeI-ClaI fragment to pET 15b.
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| Figure 2.
DNA and amino acid sequence of maize NRX. The
merged cDNA clones resulted in a composite clone 2005 bp in length. The
deduced amino acids are shown directly under the sequence. The amino
acid sequence underlined represents the specific area of the maize NRX
to which rabbit polyclonal antibodies were raised. A vertical bar
between amino acids 486 and 487 indicates where the carboxy-terminal
extension begins. Preliminary sequence information from genomic PCR
fragments suggests the location of two introns, indicated by the
darkened triangles. A potential polyadenylation is double-underlined.
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For expression of the full-length NRX protein, the coding region of Z10
was cloned into pET 15b using engineered NdeI and BamHI sites, as described above.
The full-length and the truncated expression plasmids were
established in BL21DE3, and expression of NRX was induced with isopropylthio--galactoside at a final concentration of 1 mM. After induction, the bacteria were subjected to several
freeze/thaw cycles, treated with DNase, and centrifuged to recover
the soluble protein fraction essentially according to standard
protocols (Sambrook et al., 1989). Both of these constructs resulted in
target-protein expression in the soluble and insoluble fractions. The
soluble fraction was subjected to affinity purification on an
immobilized Ni column. Inherent thrombin cleavage sites in the NRX
sequence precluded thrombin cleavage to release the His tag.
Consequently, the proteins purified from pET 15b retaining the His
leader correlated with their predicted sizes of 30 and 65 kD for NRX
and NRX, respectively (data not shown).
Polyclonal Antibody Production and Clarification
The original NRX supernatant fraction obtained from the
construct Z10-14 was subjected to Mono-Q chromatography using a linear gradient from 0 to 1.0 M NaCl in 50 mM Tris-HCl
at pH 8.0. Fractions containing the 30-kD NRX protein were
identified by electrophoresis and staining with Coomassie blue, and
were then pooled and subjected to a second round of Mono-Q
chromatography. Fractions containing NRX were pooled and subjected
to size chromatography on Sephadex-75 in 50 mM Tris HCl at
pH 8.0. Once again, fractions containing NRX were pooled and their
purity was estimated at greater than 95% by Coomassie blue staining.
These NRX fractions were used for the production of polyclonal
rabbit antibodies (Bioworld, Dublin, OH).
Clarified polyclonal antibodies were prepared by incubation of the
rabbit serum with acetone powder of whole-cell proteins isolated from
an induced BL21DE3 line transformed with pET 3Xa, which contains the T7
capsid protein (Harlow and Lane, 1988). The acetone powder preparation
was used at 1% for 30 min at 4°C, spun at 10,000g for 10 min, and then the supernatant was saved as the clarified antibody
stock.
Western Analysis
Two grams of frozen tissue of 1-week-old maize seedlings or 10-DAP
kernels were pulverized in a mortar and pestle with liquid nitrogen,
then resuspended in TBS, pH 7.6, with 1 mM EDTA and a
protease inhibitor cocktail tablet (1 873 580, Boehringer Mannheim). After spinning at 14,000 rpm for 10 min, portions of the extract supernatant were analyzed on a standard 10% acrylamide SDS
denaturing gel (Sambrook et al., 1989). The gel was
subsequently electroblotted to a nitrocellulose membrane. After
blocking in TBS with 7% dry milk powder the membrane was incubated
with the clarified anti-NRX polyclonal antibodies at a dilution of
1:30,000. The secondary antibody was used at a dilution of 1:7,000.
After washing the membrane, the SuperSignal substrate system (Pierce)
was used.
Test for TRX Activity
Both the full-length NRX protein and NRX were assayed for TRX
enzymatic activity using the insulin-disulfide reduction assay (Holmgren, 1979). The 100-µL reaction volume contained 84 mM sodium phosphate, pH 7.0, 2 mM EDTA, and
0.17 mM insulin (I 5523, Sigma). Spirulina TRX
(T 3658, Sigma) served as a positive control and BSA as a negative
control. The assay was initiated by the addition of 1 mM
DTT. Measurements were performed at 650 nm at 5-min intervals on a
spectrophotometer (model DU7400, Beckman). The cuvettes were standardized with water blanks prior to monitoring the insulin disulfide reductions.
Northern Analysis
For scrutiny of organ-specific expression of NRX mRNA, total RNA
was extracted from maize suspension-cultured cells, immature
kernels, and leaves, roots, and stems from 1-week-old seedlings using
Trizol (Life Technologies). Approximately 7 µg of total RNA was
size-fractionated in a 1.2% Mops/formaldehyde gel and transferred
to Hybond-N+ membrane (Amersham) via capillary
transfer in 20× SSC. The membrane was rinsed briefly in water, dried
at 80° for 10 min, and UV cross-linked for 2.5 min. The membrane was
hybridized to a random-prime-labeled probe prepared from an
EcoRI insert from ZmD1.
Southern Analysis
Approximately 10 µg of genomic DNA isolated from maize
suspension-cultured cells was digested with representative restriction enzymes identified in the cDNAs isolated in this study. The samples were subsequently resolved on a 1.5% agarose gel in 1× TBE and transferred to Hybond-N+ membrane via capillary
transfer in 10× SSC. The membranes were rinsed briefly in 2× SSC,
dried at 80°C for 10 min, and UV-cross-linked for 2.5 min. The same
probe prepared for the northern analysis was also used in the Southern
analysis.
Immunolocalization
Immature kernels (13 DAP) were embedded in paraffin medium
(Paraplast Plus, Sigma) as previously described (Cheng et al., 1996).
The lengthwise sections were cut at a 12-µm thickness with a rotary
microtome and mounted onto slides (Probe On Plus, Fisher Scientific)
overnight on a slide warmer set at 42°C. The paraffin medium was
removed from the sections with xylene, and the sections were rehydrated
with a graded-ethanol dilution series of 95%, 75%, 50%, and 25%.
After a brief rinse in water, the sections were incubated in PBS prior
to initiating immunogold staining as specified for the Histogold kit
(Zymed, San Francisco, CA). The primary antibody was used at a dilution
of 1:7000. A preimmune serum was included as a negative control.
Application of anti-rabbit secondary IgG antibodies conjugated to gold
particles was followed by silver enhancement to discern the specificity
of this reaction. Initially, the primary antibody incubation was
performed overnight, but a 4-h incubation interval proved satisfactory.
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RESULTS |
Identification of Maize NRX
The original NRX cDNA clone was recovered from the screening of an
expression library with monoclonal antibodies raised against a
partially purified DNA-binding activity. Rescreening the library by
hybridization and using a modified 5-rapid amplification of cDNA ends
protocol produced a set of overlapping cDNA clones that spanned the
entire coding region of NRX (Fig. 1).
The nucleotide sequence of the maize NRX cDNA composite is presented in
Figure 2. The composite sequence is 2005 nucleotides in length, with a single open reading frame that begins
with the first ATG codon at position 119 and ends with a stop codon at position 1825. There are 118 nucleotides of 5 untranslated leader, and
180 nucleotides of 3-noncoding sequence that contains a presumptive poly(A+) addition sequence and
evidence of a poly(A+) tail. The integrity of
this reconstructed contig was confirmed by preliminary genomic sequence
data and the existence of a recent homologous Arabidopsis sequence
(accession no. AC004473).
The open reading frame encodes a protein of 569 amino acids, whose
deduced sequence is also presented in Figure 2. Density-plot-matrix analysis of the composite cDNA sequence compared against itself indicates that the bulk of the sequence is composed of a repetitive structure; 3 repeats corresponding to 162 amino acids are clearly indicated by the parallel diagonal lines accompanying the central identity diagonal of Figure 3. After the
3 repeated modules, there is a unique carboxy-terminal region
corresponding to approximately 80 amino acids preceding the 3
untranslated region and the poly(A+) tail.
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| Figure 3.
Protein dot matrix reveals the multiple repeat
nature of NRX. A dot matrix from Genworks (Intelligenetics, Oxford
Molecular Group, Oxford, UK) graphically identifies the regions
of similarity repeated within the NRX sequence. The NRX amino acid
sequence was plotted against itself. Note that the main diagonal was
not eliminated and that the repetitive nature of the maize NRX is shown
as parallel dots at intervals of 162 amino acids.
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A BLAST search (Altschul et al., 1997) of nonredundant proteins
revealed homology of the repeated modules with a novel mouse NRX and
roundworm TRX entries from genomic sequence data. Figure 4 is an alignment of the three maize NRX
TRX-like tandem repeats designated a, a*, and
a, with E. coli TRX and a plant TRX-h for reference. The E. coli TRX active site WCGPC has been
modified to a WCPPC motif in each of the aligned sequences, except for the second TRX-like module of NRX, where the central PP dipeptide sequence has been maintained but the vicinal C residues are lost. The
alignment also indicates that all of the TRX-like modules from maize
NRX, the mouse module, and the roundworm sequence possess an insertion
of approximately 25 amino acids relative to the classic 12-kD TRX
(Eklund et al., 1991). This insertion sequence is relatively conserved,
with the sequences from mouse and maize maintaining as high a degree of
similarity within the inserted sequence as is noted for the modules as
a whole (Fig. 4).
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| Figure 4.
An alignment comparing the TRX-like modules of
maize NRX to its closest homologs and prototype TRXs. The repetitive
maize TRX-like modules within NRX are identified as a,
a*, and a, respectively. The mouse NRX
sequence includes only amino acids 151 to 322 for clarity. Introducing
gaps to maximize alignments readily identifies the large expansion
within mouse NRX, the C. elegans entry, and maize NRX.
The assigned accession numbers are as follows: E. coli
TRX, M54881; Arabidopsis TRX-h, Z35474; C. elegans,
Z48795; mouse NRX, X92750; and maize NRX, U90944. Identical residues
that occur in at least four entries are in bold type and are
underlined. Secondary structures from the TRX-fold substructure are
depicted above the TRX sequence as per the method of Martin (1995).
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There is a unique extension at the carboxyl end of the third TRX-like
module of maize NRX (Fig. 2). This extension has clusters of basic
residues potentially suggestive of a bipartite nuclear-localization signal, and C residues that potentially form a Zn finger.
Tissue-Specific Distribution of NRX
The clarified antibodies produced against maize NRX were used in a
western analysis of the tissue and organ-specific distribution of NRX.
The predominant cross-reacting proteins appeared to be approximately 69 kD in size (Fig. 5), which roughly
correlates with the size of 65.5 kD for the rNRX predicted from the
cDNA sequence (MacVector). NRX cross-reacting bands were abundant in extracts from kernels and suspension cells, and less-detectable NRX
amounts were observed in roots and leaves. The smaller cross-reacting bands in the gel blots likely represent proteolytic cleavage products, since recombinant, bacterially expressed NRX occasionally shows multiple bands as well (data not shown). It remains possible at this
point that related TRXs such as TRX-h may cross-react with NRX
antibodies. However, given the strict subcellular localization noted
below, this explanation for the smaller bands is considered unlikely.
The presence of a cross-reacting band larger than the rNRX may merely
represent modified NRX molecules, as there are potential glycosylation
sites in this clone. Preliminary data suggests that NRX may also be
phosphorylated.
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| Figure 5.
Western analysis. Approximately 5 µg of a crude
protein extract was loaded onto a 10% SDS-PAGE gel to resolve the
total proteins isolated from: lane 1, 10-DAP kernels; lane 2, suspension-cell culture; lane 3, epicotyl; and lane 4, roots. Lane 5 represents approximately 0.025 µg of full-length recombinant NRX.
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NRX Is Localized to the Nucleus
Figure 6 shows the cellular
localization of maize NRX within the kernel, using the same clarified
polyclonal antibodies used in the western analysis. The antibodies to
NRX clearly cross-reacted with antigens present within the nuclei of
cells in the scutellum of kernels harvested 13 DAP (Fig. 6B). Preimmune
serum used as the primary antibody showed no staining (Fig. 6A).
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| Figure 6.
NRX is present in nuclei, as detected by
immunolocalization. A, Longitudinal section through the scutellum of a
13-DAP kernel challenged with only the preimmune serum. No detection of
cross-reactivity is evident. B, Nuclei within the scutellum of a 13-DAP
kernel cross-reacting with clarified polyclonal antibodies raised
against the carboxyl end of maize NRX.
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NRX Copy Number and Gene Expression
Hybridization analysis of multiple restriction digest profiles of
maize genomic DNA resulted primarily in single bands indicative of a single-copy gene (Fig.
7). A few faintly hybridizing bands are
visible in several lanes, and the SacI digest does
show multiple hybridizing bands. However, taken together, these data
are consistent with the interpretation that NRX is a single-copy gene,
but that there may be a few related sequences in the genome of maize.
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| Figure 7.
Southern analysis. Approximately 10 µg of
genomic DNA isolated from maize suspension-cultured cells was digested
with representative restriction enzymes to examine the relative
complexity of the NRX gene. Most of the digests resulted in a single
prominent band, indicative of a single gene family, but the
SacI digest reveals several bands.
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Northern analysis demonstrated that a single 2-kb transcript hybridizes
with the NRX cDNA insert probe (Fig. 8),
indicating that the composite cDNA sequence is likely nearly
full-length. The RNA blot also demonstrates that the mRNA for NRX is
abundant in kernels and the cell-suspension culture, consistent with
the high levels of NRX protein detected by western analysis.
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| Figure 8.
RNA analysis of NRX gene expression. Approximately
7 µg of total RNA isolated from maize suspension-cultured cells,
immature kernels, and from the leaves and roots of 1-week-old maize
seedlings was size-fractionated in a 1.2% agarose Mops/formaldehyde
gel. The RNA was then transferred to Hybond-N+ membrane in
20× SSC. After UV cross-linking, the membrane was probed with the
largest EcoRI insert fragment from ZmD1. This probe
identified a transcript size of about 2 kb. The transcript appeared to
be abundant in cultures as well as kernels. The mRNA was less abundant
in leaves and roots.
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TRX Activity of NRX
Recombinant full-length NRX and the truncated NRX (containing
the intact TRX a module and the carboxy-terminal
extension) were assayed for TRX enzymatic activity by monitoring the
reduction of insulin. Spirulina TRX was included as a
positive control, whereas BSA and DTT alone were negative controls
(Fig. 9). Both the full-length NRX (at
0.13 µM) and the truncated NRX (at 0.26 µM) demonstrated significant TRX activity above
background, and the activity was similar to that of
Spirulina TRX (used at 0.50 µM) if adjusted
for the concentration of input protein. There was an obvious lag for
both forms and a corresponding slower rate of precipitation of NRX, as
expected since the concentrations were lower than the control TRX. This
phenomenon was noted by Holmgren in his classic assay (Holmgren, 1979).
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| Figure 9.
. TRX activity shown
by disulfide reduction of insulin. Approximately 8 µg of the
truncated NRX, the full-length NRX, and a representative prokaryotic
TRX (Spirulina) were assayed for TRX activity based on
ability to reduce the disulfide bonds of bovine insulin (Holmgren,
1979). TRX accelerated the reaction more dramatically than the two NRX
proteins, yet the longer delay and the corresponding slower rate of
precipitation is consistent with assays at lower concentrations. Note
that the nonenzymatic breakage with only DTT had an extremely long lag
phase, but that the same amount of precipitate was observed after
leaving the assay cuvettes for an extended period (data not shown).
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DISCUSSION |
Maize NRX is clearly a unique member of the growing superfamily of
TRX proteins. Structural and sequence similarity with the mouse NRX
suggests the name classification of NRX, which was first coined by
Kurooka et al. (1997). The presence of three TRX-like modules within
maize NRX is consistent with members of the TRX superfamily, such as
PDIs, that appear to utilize multiple TRX-like modules as a major
mechanism for structural and functional diversification. To our
knowledge, maize NRX is the first member of the superfamily to be
recognized as having three TRX-like modules in tandem, and the first
such member of the superfamily to have a putative Zn-finger module at
its carboxy terminus.
A model for the structure of maize NRX is presented in Figure
10. The first and third TRX-like
domains (a and a) are most clearly TRX like,
sharing 35% identity with each other and possessing the vicinal C
active site. The middle domain (a*) lacks the vicinal C
active site, but shares 35% identity with a and
a. The a domain has demonstrable oxidoreductase
enzymatic activity, consistent with the structural prediction of its
TRX homology. Although the entire NRX has activity as well, independent
activity of the a domain has yet to be tested, and the
a* domain is predicted to be inactive with regard to
oxidoreductase activity. The carboxy terminus of the protein
(z) consists of a putative Zn finger, but NRX has not yet
been shown to possess DNA or metal-binding capacity.
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| Figure 10.
Model for the maize NRX protein. The three
TRX-like modules are arranged in tandem and labeled as a, a*, and a.
The last module containing the putative Zn finger is labeled "z."
Limited homology with nuclear factors corresponding to specific amino
acid regions within the maize NRX are shown in boxes, and the asterisk
in HTF 10 represents additional amino acids that were omitted for
clarity. The following accession numbers were used: v-erb-A, P12891;
CCAAT-box-binding transcription factor or nuclear factor (CTF/NF)-1B2,
P17925; mastermind, M92914; HTF 10, Q05481; v-erb-A Zn finger, I57696;
Hunch ZFN, P05064; and ZNF, F14840.
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The structural organization of maize NRX addresses several issues of
structural motifs within proteins carrying multiple TRX-like modules.
The maize NRX probably arose through gene triplication, since all three
TRX-like modules (a, a*, and a) share
approximately 35% sequence identity among themselves. Even though the
a* module lacks the active site motif, it retains overall
sequence similarity such as the double Ps in the residual active site
and other conserved residues believed to be important for conferring
TRX activity and secondary structure (Eklund et al., 1991). Therefore,
the a* domain is still recognizable as a TRX-like module.
PDIs, in apparent contrast, have two TRX-like modules (a and
a) that are separated by intervening segments (b
and b) that are not overtly homologous to TRX (Darby et
al., 1996). However, recent structural studies indicate that the
PDI-b domain retains structural similarity to a TRX
fold, suggesting that all four PDI modules arose by gene duplication or
shuffling of a common ancestral TRX (Kemmink et al., 1997). Therefore,
multiplication and divergence of TRX-like modules appears to be a major
mechanism for structural and perhaps functional diversification of the
TRX superfamily of proteins.
The TRX fold represents a substructure of TRX, missing the N-terminal
-strand and -helix. Analyses of protein classes exhibiting TRX
folds notes that insertions typically are found between the -2
strand and -2 helix (Martin, 1995). Using the E. coli TRX sequence as a reference for secondary structures, the largest insertions in the maize NRX-alignment figure (Fig. 4) occur between the
secondary structures -2 and -2 within the TRX-fold substructure. Sequence similarity and oxidoreductase activity for mouse and maize NRX
is suggestive of such TRX folds. In addition, TRX-fold-containing proteins such as glutathione peroxidase and DsbA (the bacterial equivalent of PDI) have even larger insertions in this region, 40 and
76 amino acids, respectively (Martin, 1995), compared with the 23 to 24 amino acids found in maize NRX.
Intron capture has been invoked to explain the expanded TRX module in
the roundworm TRX-like entry (Sahrawy et al., 1996). Although there
is a intron at the end of this substantial insertion in the roundworm
sequence entry (Z48795), preliminary genomic sequence information from
the maize NRX does not show an intron here. Furthermore, scrutiny of
the mouse and maize modules suggests conservation of the amino acids
within the region of this insertion (see Fig. 4). This sequence
conservation among vastly divergent eukaryotic species suggests that
intron capture is an unlikely explanation for the presence of these
insertions, as it is very unlikely that plants and animals would have
retained enough sequence identity within introns such that their
parallel capture would have produced the similarity of amino acid
sequence within the insertion. It is more likely that the inserted
sequence predates the radiation of plants and animals and is retained
because of functional significance.
Several members of the TRX superfamily have additional modules at the
carboxy terminus. The mouse NRX and PDIs have carboxyl extensions, but
those extensions are not similar in sequence or potential structure to
the Zn finger of maize NRX. Recently, however, a novel E. coli TRX sequence was reported to have an extension containing
extra C residues in a proper sequence context to form a putative Zn
finger, but this extension is located at the amino terminus
(Miranda-Vizuete et al., 1997). The maize NRX carboxy terminus has
clustering of basic residues suggestive of a bipartite nuclear
localization motif, and sequence analysis (Lupas, 1996) hints at a
potential for coil-coil interaction in this region. The strongest
coil-coil potential exists near amino acids 471 to 493, which also has
homology to a domain within a recently reported predicted Arabidopsis
receptor-kinase-like protein (accession no. AL021633).
The presence within the nucleus of a multiple-TRX modular protein with
a putative Zn finger offers intriguing possibilities for the regulation
of transcription factors by alteration of their redox state. The number
of transcription factors known to be influenced by changes in their own
redox state is fairly large and increasing. TRX was shown early on to
be associated with the replication machinery as an integral part of T7
polymerase, and even E. coli TRX had been noted to be
associated with the nucleoid region of some E. coli cells
(Holmgren, 1979; Nygren et al., 1981). The potential association of NRX
with critical nuclear events is further enhanced by the presence within
NRX of sequence elements that bear limited homology to the
CCAAT-box-binding transcription factor or nuclear factor, mastermind,
and v-erb-A (Fig. 10). Mastermind accumulates in the nucleus and is an
important neurogenic locus. The limited homology with v-erb-A is also
found in the thyroid hormone receptor -2. Representative Zn fingers
also depicted in Figure 10 hint at the putative maize NRX Zn finger as
being similar to the human HTF 10 sequence, with the exception of
having the C pairs more closely associated. Elucidation of these
potential functional associations awaits further characterization of
NRX and other NRX-like proteins.
| |
FOOTNOTES |
*
Corresponding author; e-mail robferl{at}nervm.nerdc.ufl.edu; fax
1-352-392-6479.
Received April 13, 1998;
accepted August 5, 1998.
1
This research was supported by the National
Institutes of Health (grant no. GM40061 to R.J.F.). This manuscript is
journal series no. R-06452 of the Florida Agricultural Experiment
Station.
| |
ABBREVIATIONS |
Abbreviations:
DAP, days after pollination.
NRX, nucleoredoxin.
PDI, protein disulfide isomerase.
TRX, thioredoxin.
| |
ACKNOWLEDGMENTS |
The authors are especially grateful to Prem Chourey, who
graciously provided the maize kernels for sectioning as well as the expertise for preparing these tissues. We also thank Susan Carlson, who
patiently assisted in this preparation as well as generously guiding us
in immunostaining and subsequent photo documentation. In addition, we
thank Ernesto Almira and Savita Shankar for facilitating the automated
sequencing through the ICBR facility at the University of Florida.
| |
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Abstract
Introduction