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.

Subcellular localization of Dhh1, Pat1 and polysomal complexes during exponential growth and the growth retardation phase Dhh1 and Pat1 have been shown to be capable of acting, at least under certain conditions, as translational repressors (3). However, knockouts of these genes have not been observed to lead to enhanced protein synthesis rates (4), thus indicating that we have yet to understand the role of these proteins in controlling global translation and thus cell growth. In order to throw light on this poorly understood area, we used fluorescent tags to label Dhh1 and Pat1 and then compared their subcellular distributions with those of GFP-tagged 40 S and 60 S ribosomal subunits under different growth conditions. In the first instance, we chose to use the tetracysteine motif (TCM) tag combined with biarsenical dyes (22, 23) to label Dhh1 and Pat1. The TCM fusion constructs used in this work were chromosomally integrated behind the respective natural promoters (see 'Materials and Methods' section) so that the intracellular levels of the encoded fusion proteins were the same as the normal endogenous levels of the non-fused proteins.