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.

However, we also observed that Dhh1 and Pat1 colocalize more extensively with ribosomes downstream of the diauxic shift; this is again illustrated by single Z-section images in Figure 1C . In the merged single section images, the overlap between the GFP-and TCM-tagged proteins is readily identified as yellow patches in the cells. Again, switching the tags between ribosomal subunits and the proteins under study did not alter the observed respective subcellular distributions (Supplementary Figure S3 shows single Z-section images). We also observed that eIF4A, eIF4E, eEF3 and eRF1 remain relatively evenly distributed throughout the cytoplasm and demonstrate only very limited co-localization with Dhh1 and Pat1 in both exponential growth and in the ethanol respiration phase (Supplementary Figures S4 and S5 show single Z-section fluorescence images). It has previously been reported that eIF4E, eIF4G and Pab1 are found in P bodies (24) and also in what are believed to be distinct bodies that form upon prolonged glucose depletion (EGP bodies, 25) . The shift in distribution of Dhh1 and Pat1 that we have observed evidently allows these proteins to share the subcellular regions in which most of the ribosome pool resides. DAPI staining was used to locate the positions of the nuclei in glucose-fermenting exponentially growing cells and in cells that have undergone the diauxic shift (Supplementary Figure S6) , revealing that at least 90% of the Dhh1 and Pat1 TCM-tag fusions, labelled with ReAsH, are not in the nucleus.