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.

Focusing first on cells growing exponentially on glucose, we made an unexpected observation: the pool of ribosomes [large (Rpl25a) and small (Rps2) subunits] was partly segregated from Dhh1 and Pat1. This is most evident in single Z-section (not 3D) images in Figure 1A . In controls, western blot analysis of polysomal gradient fractions revealed that the GFP-fused ribosomal subunit proteins were exclusively associated with assembled ribosomal subunits (data not shown). In order to control for any potential influence of the chosen protein tagging strategy, we repeated this experiment using GFP-tagged Dhh1 and Pat1 together with TCM-tagged ribosomes ( Figure 1B ; fluorescence labelling controls are shown in Supplementary Figure S1 ). The advantage of utilizing both tagging procedures is that the 13mer TCM tag is far smaller than GFP, so that any size-related effects of protein tagging could be controlled for by this approach. Moreover, by imaging live cells, we avoided the artefacts that are often associated with microscopy of fixed cells. We next investigated whether this apparent segregation was maintained after cells have undergone the diauxic growth shift from glucose fermentation to ethanol oxidation. It is important to note that we imaged cells prior to the stationary phase since, by definition, in this latter state large numbers of cells are dying (Supplementary Figure S1 ). We observed the previously reported (15) progressive migration of Dhh1 and Pat1 into P bodies in response to the diauxic shift, although not all of the intracellular Dhh1 and Pat1 locates to the P bodies (Supplementary Figure S2 shows 3D projections from serial Z-axis images).