Secretory pathway genes assessed by high-throughput microscopy and synthetic genetic array analysis

Abstract

We developed a procedure for automated confocal microscopy to image the effect of the non-essential yeast gene deletion set on the localisation of the plasma membrane GFP-labelled protein Mrh1p-GFP. To achieve this it was necessary to devise an expression system expressing Redstar2 RFP-fluorescence specifically in the nucleus, mCherry RFP at a lower intensity in the cytoplasm and Mrh1p-GFP in the plasma membrane. This fluorescence labelling scheme utilising specifically designed image analysis scripts allowed automated segmentation of the cells into sub-regions comprising nuclei, cytoplasm and cell-surface. From this high-throughput high content screening approach we were able to determine that gene deletions including emc1Δ, emc2Δ, emc3Δ, emc4Δ, emc5Δ and emc6Δ, caused intracellular mislocalisation at the ER of a plasma membrane protein Mrh1p-GFP. CPY processing patterns were unaffected in these mutants and collectively our data suggest a transport role for the EMC genes within the early secretory pathway. HAC1 is central to the unfolded protein response (UPR) and in its absence, i.e. the absence of UPR, emc1Δ-, emc3Δ-, emc4Δ-, emc5Δ-hac1Δ double mutants were specifically hypersensitive to ER-stress (tunicamycin) lending credence to the usefulness of the high content microscope screening for discovery of functional effects of single mutants.

SCS3 and YFT2 Link Transcription of Phospholipid Biosynthetic Genes to ER Stress and the UPR

“…Thus, the strong aggravating phenotype of the sec13-1 scs3 Δ yft2 Δ strain may be explained by hypersensitivity to secretory stress alone or in combination with altered phospholipid composition. Indeed, deletion of SCS3 has been reported to cause elevated UPRE-reporter gene expression [86], [87] and to confer hypersensitivity to tunicamycin in UPR-defective strains, consistent with elevated levels of ER stress [88]. These observations prompted a closer evaluation of SCS3 and YFT2 function during induction and attenuation of the UPR.…”

A yeast phenomic model for the gene interaction network modulating CFTR-ΔF508 protein biogenesis

“…Our interpretation of these data is that EMC and Sec61 act in a functionally distinct pathway from ERAD, pathways that can buffer loss of one another [5,76]. Other evidence suggesting a role for the EMC in the early secretory pathway comes from a high content microscopy screen, which discovered loss of the EMC causes increased ER retention of the Mrh1-GFP fusion protein [77]. Importantly, we note that the role of the EMC and other secretory protein biogenesis network factors appears cargo-specific, since other factors that were found in the Mrh1-GFP screen exerted qualitatively different effects in our Yor1-ΔF screen [77].…”

“…Other evidence suggesting a role for the EMC in the early secretory pathway comes from a high content microscopy screen, which discovered loss of the EMC causes increased ER retention of the Mrh1-GFP fusion protein [77]. Importantly, we note that the role of the EMC and other secretory protein biogenesis network factors appears cargo-specific, since other factors that were found in the Mrh1-GFP screen exerted qualitatively different effects in our Yor1-ΔF screen [77]. From a detailed comparison of our screen with the list of genes described by Bircham et al .…”

Functional genomics in the study of yeast cell polarity: moving in the right direction

“…To date, image-based screens using the yeast deletion and other arrayed collections have collected static images in a single focal plane [53,[90][91][92][93][94][95][96][97][98]. However, cell polarity is highly dynamic, and future analyses will involve the high-throughput acquisition of three-and fourdimensional spatial and temporal image sequences, using multiple fluorescent markers to highlight numerous compartments simultaneously.…”

“…So far, the GFP library has seen limited use for exploring cell polarity pathways-one screen identified proteins that localize to the mating projection in pheromone-treated cells [89]. A more common approach has been to study changes in the localization of cell polarityspecific proteins fused to a fluorescent moiety in the deletion collection or in the context of gene overexpression [53,[90][91][92][93][94][95][96][97][98]. For instance, the deletion mutant array was screened for mislocalization of modified Snc1-GFP, yeast synaptobrevin, to identify novel regulators of its internalization [53].…”