<em>Zea mays</em> Taxilin Protein Negatively Regulates Opaque-2 Transcriptional Activity by Causing a Change in Its Sub-Cellular Distribution

<div><p><em>Zea mays</em> (maize) Opaque-2 (ZmO2) protein is an important bZIP transcription factor that regulates the expression of major storage proteins (22-kD zeins) and other important genes during maize seed development. ZmO2 is subject to functional regulation through protein-protein interactions. To unveil the potential regulatory network associated with ZmO2, a protein-protein interaction study was carried out using the truncated version of ZmO2 (O2-2) as bait in a yeast two-hybrid screen with a maize seed cDNA library. A protein with homology to Taxilin was found to have stable interaction with ZmO2 in yeast and was designated as ZmTaxilin. Sequence analysis indicated that ZmTaxilin has a long coiled-coil domain containing three conserved zipper motifs. Each of the three zipper motifs is individually able to interact with ZmO2 in yeast. A GST pull-down assay demonstrated the interaction between GST-fused ZmTaxilin and ZmO2 extracted from developing maize seeds. Using onion epidermal cells as <em>in vivo</em> assay system, we found that ZmTaxilin could change the sub-cellular distribution of ZmO2. We also demonstrated that this change significantly repressed the transcriptional activity of ZmO2 on the 22-kD zein promoter. Our study suggests that a Taxilin-mediated change in sub-cellular distribution of ZmO2 may have important functional consequences for ZmO2 activity.</p> </div

ZmTaxilin can repress the transcriptional activity of ZmO2.

<p>(<b>A</b>) Structure of effecter and reporters. (<b>B</b>) GUS/luciferase values of different reporter and effecter combinations. The values are the averages with SD of three independent experiments, after normalisation to the internal control. Statistical significance between YFP-O2 and YFP-O2+CFP-Taxilin was calculated using a two-tailed T-test. *p≤0.05.</p

ZmTaxilin and ZmO2 interact in a GST pull-down assay.

<p>(<b>A</b>) Western blot detection of the GST pull-down sample with a GST antibody. The kernel sample was a pool of equal amounts of RNA from different developmental stages between 3 and 36 days after pollination (DAP). (<b>B</b>) Western blot detection of the GST pull-down sample with the ZmO2 antibody. Lane 1 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing the GST-Taxilin protein and the maize seed protein containing ZmO2. Lane 2 in (<b>A</b>) and (<b>B</b>): <i>E. coli</i> lysate containing GST and maize seed protein containing ZmO2. The expected molecular weight of the GST-Taxilin fusion protein, the GST tag and ZmO2 are 75.397, 27.895 and 47.075 kDa, respectively. The apparent molecular weight of the ZmO2 protein was approximately 68–72 kDa.</p

<i>ZmTaxilin</i> clones identified by yeast two-hybrid.

<p><i>ZmTaxilin</i> clones identified by yeast two-hybrid.</p