title: 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.
authors: Department of Biological Sciences, Victoria University of Wellington, Kelburn Parade, Wellington, New Zealand.
authors: Peter W
authors: PW
authors: Bircham
authors: David R
authors: DR
authors: Maass
authors: Christina A
authors: CA
authors: Roberts
authors: Poh Y
authors: PY
authors: Kiew
authors: Yee S
authors: YS
authors: Low
authors: Manivannan
authors: M
authors: Yegambaram
authors: James
authors: J
authors: Matthews
authors: Cameron A
authors: CA
authors: Jack
authors: Paul H
authors: PH
authors: Atkinson
mesh_terms: D002462:Cell Membrane / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins