title: A high-content screening microscopy approach to dissect the role of Rab proteins in Golgi-to-ER retrograde trafficking.
abstract: Here, we describe a high-content microscopy-based screen that allowed us to systematically assess and rank proteins involved in Golgi-to-endoplasmic reticulum (ER) retrograde transport in mammalian cells. Using a cell line stably expressing a GFP-tagged Golgi enzyme, we used brefeldin A treatment to stimulate the production of Golgi-to-ER carriers and then quantitatively analysed populations of cells for changes in this trafficking event. Systematic RNA interference (RNAi)-based depletion of 58 Rab GTPase proteins and 12 Rab accessory proteins of the PRAF, YIPF and YIF protein families revealed that nine of these were strong regulators. In addition to demonstrating roles for Rab1a, Rab1b, Rab2a, and Rab6a or Rab6a' in this transport step, we also identified Rab10 and Rab11a as playing a role and being physically present on a proportion of the Golgi-to-ER tubular intermediates. Combinatorial depletions of Rab proteins also revealed previously undescribed functional co-operation and physical co-occurrence between several Rab proteins. Our approach therefore provides a novel and robust strategy for a more complete investigation of the molecular components required to regulate Golgi-to-ER transport in mammalian cells.
authors.affiliation: School of Biology and Environmental Science and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: George
authors.initials: G
authors.lastname: Galea
authors.affiliation: School of Biology and Environmental Science and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Bexiga
authors.affiliation: School of Biology and Environmental Science and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: Angela
authors.initials: A
authors.lastname: Panarella
authors.affiliation: School of Biology and Environmental Science and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland.
authors.forename: Elaine D
authors.initials: ED
authors.lastname: O'Neill
authors.affiliation: School of Biology and Environmental Science and UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland jeremy.simpson@ucd.ie.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Simpson
mesh_terms: D001681:Biological Assay; D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008853:Microscopy / Q000379:methods*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D015203:Reproducibility of Results; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
chemical_list: D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D020691:rab GTP-Binding Proteins
keywords: High-content screening; Rab proteins; Retrograde traffic