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.

The co-localization of Dhh1 and Pat1 with the translation machinery raised the question as to what interactions underpin the observed association with the translation machinery and, most importantly, how these interactions change in response to changes in the cellular environment. We therefore used pull-down experiments to investigate whether components of the translation machinery could be identified as interaction partners of Dhh1 and Pat1 under our experimental conditions, thus defining key interactions at the interface between translation and mRNA decay. TAP-tagged Dhh1 was utilized in this study as bait in pull-down experiments ( Figure 4) . These data reveal that, during exponential growth, Dhh1 is complexed with Pat1, the elongation factor eEF1A and several ribosomal proteins, whereas after the diauxic growth shift not only does Dhh1 retain its association with Pat1 and eEF1A but it also becomes complexed with Ded1 and eIF4A. To determine if this co-precipitation is mRNA dependent, the pull-down experiment was repeated as before or with cell extracts pre-treated with RNase A and the resultant eluents analysed by western blotting ( Figure 4B ). RNase A cleaves 3 0 of C and U residues and will therefore break down the body of each mRNA into small fragments. This indicates that the Ded1, eEF1A and eIF4A associate with Dhh1 in an RNA-independent manner and again suggests that the association of Dhh1 with Ded1 and eIF4A is enhanced in response to the diauxic growth shift. We also explored whether Dhh1 and Pat1 are associated with ribosomal preparations obtained from yeast. Partially purified ribosomes were subjected to washes with 500 mM KCl or 1 M KAc to generate protein-containing supernatants (Supplementary Figure S7 , supernatants S2 and S3, respectively) and a ribosomal pellet (P), and the proteins in these fractions were identified using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS; see 'Materials and Methods' section, Supplementary Figures S7 and S8 and Figure 5 ). Exponentially modified Protein Abundance Index (emPAI) abundance scores reflect the number of peptides observed per protein ( Figure 5A) (21, 26, 27) . We also determined the MASCOT values for the same data sets; these correlate well (with one significant outlier) with the emPAI scores ( Figure 5B ). These label-free quantitation data indicate that Dhh1 and Pat1 retain their association with ribosomes after washing with 500 mM KCl, but that both proteins are released (into supernatant S3) upon treatment with 1 M KAc. Interestingly, a number of translation initiation and elongation factors (e.g. eIF1A, eIF4A and eEF1A, eEF3, respectively), and Ded1, are found in all fractions, whereas eRF1 and the ribosomal proteins are identified primarily in the 1 -M KAc supernatant (S3) and the ribosomal pellet (P). Overall, these data suggest that Dhh1 and Pat1 can be relatively stably associated with ribosomes, although they do not tell us whether other factors are required for this association.