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.

Visual assessment of the subcellular distributions of proteins and complexes can be misleading, and we therefore performed quantitative fluorescence intensity analysis of the distribution of Dhh1/Pat1, ribosomes and translation factors throughout individual yeast cells. Cells were imaged in three dimensions, whereby a minimum of 50 serial sections were collected at 0.1-micron intervals and, once deconvolved, the Manders Intersection coefficients (i) were calculated for each tagged protein combination (e.g. Dhh1-TCM and Rpl25-GFP, Pat1-TCM and Rps2-GFP etc.) in either exponential growth or subsequent to the diauxic shift. These analyses allow the degree of intersection/overlap of the red (TCM-ReAsH) signal with the green (GFP) signal to be displayed graphically (Figure 2 ). These 3D, whole-cell distribution analyses reveal that during exponential growth the majority of Dhh1 and Pat1 ($75-80% of the imaged signal) is segregated from ribosomal subunits and translation factors. Upon diauxic growth shift, Dhh1 and Pat1 distribution relative to ribosomes undergoes a striking reorganization and the majority of Dhh1 and Pat1, presumably in the context of P-bodies ( Figure 1C) , intersects with ribosomal proteins. Interestingly, this growth-phase-specific differential distribution is not mirrored to the same extent when comparing Dhh1/Pat1 and translation factors. However, Dhh1 and Pat1 do show a small relative increase in signal overlap with several factors after the diauxic shift (Figure 2 and Supplementary Figure S4) .