DOI: 10.5061/dryad.20dv6sg
title: Data from: Collagen secretion screening in Drosophila supports a common secretory machinery and multiple Rab requirements

description: Collagens are large secreted trimeric proteins making up most of the
 animal extracellular matrix. Secretion of collagen has been a focus of
 interest for cell biologists in recent years because collagen trimers are
 too large and rigid to fit into the COPII vesicles mediating transport
 from the endoplasmic reticulum (ER) to the Golgi. Collagen-specific
 mechanisms to create enlarged ER-to-Golgi transport carriers have been
 postulated, including cargo loading by conserved ER exit site (ERES)
 protein Tango1. Here, we report an RNAi screening for genes involved in
 collagen secretion in Drosophila. In this screening, we examined
 distribution of GFP-tagged Collagen IV in live animals and found 88 gene
 hits for which the knockdown produced intracellular accumulation of
 Collagen IV in the fat body, the main source of matrix proteins in the
 larva. Among these hits, only two affected collagen secretion
 specifically: PH4αEFB and Plod, encoding enzymes known to mediate
 posttranslational modification of collagen in the ER. Every other
 intracellular accumulation hit affected general secretion, consistent with
 the notion that secretion of collagen does not use a specific mode of
 vesicular transport, but the general secretory pathway. Included in our
 hits are many known players in the eukaryotic secretory machinery, like
 COPII and COPI components, SNAREs and Rab-GTPase regulators. Our further
 analysis of the involvement of Rab-GTPases in secretion shows that Rab1,
 Rab2 and RabX3, are all required at ERES, each of them differentially
 affecting ERES morphology. Abolishing activity of all three by Rep
 knockdown, in contrast, led to uncoupling of ERES and Golgi. We
 additionally present a characterization of a screening hit we named
 trabuco (tbc), encoding an ERES-localized TBC domain-containing Rab-GAP.
 Finally, we discuss the success of our screening in identifying secretory
 pathway genes in comparison to two previous secretion screenings in
 Drosophila S2 cells.
description: Collagen IV (Vkg.GFP)
 primary screeningConfocal micrographs
 (TIFF image files) of Drosophila L3 fat body, corresponding to Fig. S1 in
 Ke et al., 2018. The compressed folder contains images for the wild type
 (WT_vkgGFP.tif) and the 88 intracellular retention hits in the primary
 Collagen IV secretion screening (gene symbol_vkgGFP.tif). Apart from
 Collagen IV (Vkg.GFP) in green, plasma membrane is shown in red (myr.RFP)
 and nuclei in blue (DAPI). Images were acquired in a Zeiss LSM780
 microscope with a 63x oil immersion objective.vkgGFP.rarPerlecan (Trol.YFP) secondary screeningConfocal micrographs (TIFF image files) of Drosophila L3 fat body, corresponding to Fig. S2 in Ke et al., 2018. The compressed folder contains images for the wild type (WT_trolYFP.tif) and the Perlecan secondary screening of the 88 intracellular retention hits in the primary Collagen IV screening (gene symbol_trolYFP.tif). Apart from Perlecan (Trol.YFP) in green, plasma membrane is shown in red (myr.RFP) and nuclei in blue (DAPI). Images were acquired in a Zeiss LSM780 microscope with a 63x oil immersion objective.trolYFP.rarGeneral secretion (secr.GFP) secondary screeningConfocal micrographs (TIFF image files) of Drosophila L3 fat body, corresponding to Fig. S3 in Ke et al., 2018. The compressed folder contains images for the wild type (WT_secrGFP.tif) and the general secretion secondary screening of the 88 intracellular retention hits in the primary Collagen IV screening (gene symbol_secrGFP.tif). Apart from the general secretion marker (secr.GFP) in green, plasma membrane is shown in red (myr.RFP) and nuclei in blue (DAPI). Images were acquired in a Zeiss LSM780 microscope with a 63x oil immersion objective.secrGFP.rar