A novel nuclear member of the thioredoxin superfamily

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.

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 λ-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.

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.

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.

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).

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.

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.

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.

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.

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).

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.