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 observed co-localization does not tell us whether Dhh1 and Pat1 associate with particular subclasses of ribosomes, and we therefore investigated whether the chromosomally encoded TCM fusions become associated with actively translating, or non-translating, ribosomes under these conditions. Western blotting of fractions generated by sucrose gradient fractionation of cell extracts revealed that Dhh1 and Pat1 are not present in the polysomal fractions during exponential growth, but do appear in the polysomal fractions when cells undergo the diauxic shift ( Figure 3B ). That Pat1 is limited to monosomal fractions in polysomal gradients prepared from exponentially growing cells was also observed previously (5) . The observed co-fractionation effects might conceivably have been attributable to Dhh1 and Pat1 being incorporated into P bodies that run in the same fractions as polysomes. We therefore performed two types of control experiment ( Figure 3C ). The addition of puromycin to inhibit translation drastically reduced the content of polysomes in the higher mass fractions and also shifted the distribution of Dhh1 and Pat1 back into the lower mass fractions, just as would be expected if these two proteins are associated with polysomal complexes. Similarly, the addition of RNase A, which cleaves single-stranded RNA 3 0 of C and U residues, led to both the collapse of polysomes and the redistribution of Dhh1 and Pat1 into the lower mass monosomal and ribonucleoprotein fractions. Overall, these data suggest that the co-localization observed in diauxic growthshifted cells reflects genuine association between Dhh1, Pat1 and translationally active polysome complexes.