Diauxic shift-dependent relocalization of decapping activators Dhh1 and Pat1 to polysomal complexes

Drummond, Sheona P.; Hildyard, John; Firczuk, Helena; Reamtong, Onrapak; Li, Ning; Kannambath, Shichina; Claydon, Amy J.; Beynon, Robert J.; Eyers, Claire E.; McCarthy, John E. G.

While detailed characterization of the mechanistic details underpinning the re-localiization of Dhh1 and Pat1 within the cell is beyond the scope of the present article, we have obtained evidence that suggests that eIF4A, and/ or translation initiation, promotes this re-localization (compare Figure 6 and Supplementary Figure S10) . Thus eIF4A and the decapping activators may act synergistically to promote access to actively translating polysomal complexes. Earlier studies have indicated that eIF4A (in comparison to Ded1) is not effective in promoting the long-term scanning stability of ribosomal pre-initiation complexes that have to negotiate long Relative expression levels of eIF4A were determined by labelling with a FITC-conjugated secondary antibody, followed by visualization with a Typhoon Biomolecular Imager (GE Healthcare) and analysis using ImageQuant software, using hexokinase as a loading control. The far-left lane (labelled DHH1::GFP) shows the expression levels of hexokinase and eIF4A in a control strain in which the DHH1::GFP fusion is transcribed from the natural chromosomal promoter. The other three lanes show, as labelled, 95% expression (0 ng/ml DOX-the promotor substitution induces a 5% decrease in expression level compared to wild-type), 70% (5 ng/ml DOX) and 50% (10 ng/ml DOX). (B) Cells were treated as in panel A and then grown to an OD 600 = 0.5 (exponential) or OD 600 = 2.0, were visualized on a Deltavision microscope and then 3D projections were generated from 50 serial Z-axis images collected at 0.1-micron intervals. (C) Cells expressing 95, 70 or 50% of the wild-type level of eIF4A were harvested during exponential (left) or retardation phase (diauxic growth shift; right) growth and extracts from these cells were then analysed by polysomal gradient fractionation. Corresponding polysomal gradient fractions were collected and proteins resolved by SDS-PAGE and probed using anti-GFP antibodies to determine the distribution of Dhh1-GFP. 5 0 -UTRs (33, 34) . In contrast, this result may have identified a more significant role for this initiation factor in which its remodelling capability is brought to bear to enable other proteins access to translationally active mRNPs. In addition, we have found evidence of changes in the interactome of Dhh1 triggered by the diauxic growth shift that are likely to reflect corresponding alterations in mRNP structure. This also suggests that there is remodelling of the translating mRNP downstream of the diauxic shift, allowing closer association of Dhh1 and Pat1. The results of the pull-down experiments suggest that this remodelling may be promoted or supported by the DEAD-box helicases Ded1 and eIF4A which, together with Dhh1 (and possibly Pat1), become recruited into the remodelled mRNP formed in response to diauxie. That a group of helicases is involved in this remodelling process is consistent with the perceived general role of this class of proteins in the cell (35) . The closer association of Dhh1, Pat1, Ded1 and eIF4A in a remodelled mRNP may possibly serve to partially inhibit the positive roles played by Ded1 and eIF4A in the fully active process of translation initiation. Overall, the results presented here indicate that future work should focus on further investigation of the potential role of at least these four proteins in the broader context of mRNP restructuring as a means of modulating the translational activity of polysomes in response to changes in the cell environmen