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 Golgito-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.

Spatial and Functional Aspects of ER-Golgi Rabs and Tethers

“…Also, the dynamics of Rab2 has not been recorded in living cells. Interestingly, however, Rab2A was recently shown by LM to overlap with ERGIC-53 and Rab1B (Sugawara et al, 2014 ; Galea et al, 2015 ), confirming its IC localization.…”

“…Of note, although COPI coats function in retrograde trafficking, a recent study re-emphasized their additional role in anterograde transport at the Golgi level (Park et al, 2015 ). Finally, an LM-based screen identified the Rab1 and Rab2 isoforms as key regulators of retrograde transport of Golgi enzymes to the ER in BFA-treated cells (Galea et al, 2015 ), further demonstrating their requirement for bidirectional ER-Golgi communication.…”

“…Besides ER-Golgi trafficking, the isoforms of Rab1 and Rab2 are also important for Golgi biogenesis (Wilson et al, 1994 ; Haas et al, 2007 ; Rendón et al, 2013 ; Liu and Storrie, 2015 ), indicating that the two prosesses are intimately coupled. Accordingly, their knock-down results in the fragmentation of the Golgi ribbon (Galea et al, 2015 ). Interestingly, a recent study showed that the Golgi fragmentation phenotype could be induced by single knock-down of each of the four isoforms.…”

High-content analysis of Rab protein function at the ER-Golgi interface

“…In order to further investigate this, we have recently reported the effects of systematic protein depletion of the Rab protein family in the context of transport pathways exiting the Golgi complex, particularly toward the ER. 21 This study provides the first systematic view of the key Rab molecules operating at the ER-Golgi interface with new implications for how Golgi architecture is maintained.…”

Epistatic Interplay between Type IV Secretion Effectors Engages the Small GTPase Rab2 in the Brucella Intracellular Cycle

“…RicA alters host secretory traffic and Golgi apparatus morphology. RicA was identified as binding Rab2 in vitro (14), which regulates Golgi apparatus-ER retrograde traffic (24)(25)(26), and yet whether RicA activity impairs Rab2-dependent secretory transport has not been established. We first verified the occurrence of RicA-Rab2 interactions in mammalian cells, by coimmunoprecipitation of ectopically expressed myc-tagged RicA with wild-type, dominant-negative (Rab2 S20N ) or constitutively active (Rab2 Q65L ) alleles of green fluorescent protein (GFP)-tagged Rab2 in HeLa cells ( Fig.…”