A genome-wide RNAi screen for proteins required for secretion. (A) Schematic diagram of the reporter construct used to generate the stable S2 cell line used for the screen. The metallothionein promoter and the BiP signal peptide are from Drosophila. (B) Summary of screening protocol. Secretory pathway function was quantified by determining the proportion of total luciferase activity in the well that was in the medium fraction (i.e. A/A+B). (C) Distribution of the functions of the 108 hits from the screen (for more details see Supplementary Table 1).

Re-screen of potential hits from the genome-wide screen. Secretion of luciferase from S2 cells treated with dsRNA corresponding to the indicated genes. Secretion was quantified as in Figure 1 (i.e. the proportion of total activity present in the well that was in the medium), and normalised with respect to the GFP control included with each set (means of triplicates, error bars indicating standard deviation). Of the ‘known exocytosis' hits CG2023, CG5484, and CG7039 are the Drosophila orthologues of Sec20, Yif1, and ARFRP1 respectively. In total 13 of the hits for known components of exocytosis were re-screened with different amplicons and all were positive again, indicating the robustness of the assay system.

Localisation of tagged forms of the proteins encoded by selected genes identified in the screen. (A, B) Confocal micrographs of S2 cells transfected with plasmids expressing the indicated genes tagged at the C terminus with the V5 epitope. Cells were labelled for the V5 tag and the Golgi marker dGM130, or the ER exit site component dSec16. Scale bars 5 μm (A) or 1 μm (B). (C) Summary of the localisation data in (A, B) for the eight selected genes along with the gene names for their human orthologues.

Effect of RNAi against CG14199/kish and gryzun on CD8-GFP expression in the Drosophila salivary gland. (A, B) Confocal micrographs of larval L3 salivary glands expressing CD8-GFP and stained for the Golgi marker dGolgin-245. The flies either express no dsRNA or express a dsRNA against the indicated gene in the salivary gland using the Gal4 system. In the absence of dsRNA, CD8-GFP accumulates as expected on the apical plasma membrane that lines the secretory duct (arrowhead in panel B). Nuclei are the very large black holes visible in the cross sections and some of the horizontal sections. For CG14199 and gryzun z-stacks of several salivary glands were analysed for each, with the same phenotypes observed in all cases, including loss of the concentration of the CD8-GFP reporter on the apical membrane. The images are taken at the same settings so that GFP levels can be compared directly. Scale bars=10 μm.

C4orf41 depletion blocks constitutive secretion from mammalian cells. (A) siRNAs against C4orf41, TMEM167A, TMEM167B, and syntaxin 5 (STX5) were transfected into a HeLaM-derived cell line (C1) that expresses a regulated secretory reporter. At 4 days after RNAi treatment, secretion of the GFP-FM4-hGH reporter was induced by adding the aggregation preventing small molecule AP21998. The fluorescence remaining in the cells was determined at the start and after 80 min using flow cytometry. The result is expressed as the proportion of material that remains in the cell (error bars show fractional deviation of triplicate data points, and the experiment was repeated twice). To confirm that the C4orf41 secretion phenotype was genuine and not an off-target effect, the C4orf41 siRNA pool was deconvoluted and three out of the four oligonucleotides found to give a substantial block in secretion. (B) Confocal time-lapse imaging of the release of the GFP-FM4-hGH reporter from C1 cells that had been mock-treated, or with siRNAs for C4orf41. Release was initiated by the addition of AP21998 after 1 min, with frames shown for the indicated time points (minutes). There is a low level of basal secretion that results in GFP accumulation on the surface at cell contact sites (asterisks at 27.2 min). In mock-treated cells the material released from the ER enters the Golgi (arrowheads at 27.2 min) and then leaves. In C4orf41 siRNA-treated cells the reduction in basal secretion causes an increase in ER labelling at the start of the experiment. Nonetheless, a substantial fraction of the material leaves the ER and accumulates in scattered structures in the cytoplasm. The full time series are in Supplementary Movies 1 and 2.

Depletion of C4orf41 fragments the Golgi ribbon and prevents secretory cargo moving beyond the Golgi fragments. (A) Wide-field micrographs of HeLaM cells treated with the indicated siRNAs and stained for the Golgi markers GM130 or TGN46, and with DAPI to visualise the nucleus (blue). Quantitation of mock-, TMEM167-, and C4orf41-treated cells revealed perturbation of GM130 in 0, 0, and 93% respectively (n=70), and of TGN46 in 0, 5, and 95% respectively (n=110). (B) Wide-field micrographs of C1 cells treated with siRNA against C4orf41 and then fixed 80 min after induction of the release of GFP-FM4-hGH from the ER as in Figure 4. The cells were labelled with the indicated markers for the cis-Golgi (GM130), the trans-Golgi (golgin-245), and lysosomes (Lamp II). To confirm the Golgi fragmentation phenotype, we performed each staining experiment with two different oligonucleotides against C4orf41, and representative results are shown.

Analysis of the yeast and mammalian orthologues of CG14199/Kish. (A) An alignment of CG14199/Kish with orthologues from the indicated species (Hs, Homo sapiens; Xl, Xenopus laevis; Sc, S. cerevisiae; At, Arabidopsis thaliana; Dd, Dictyostelium discoideum; Ce, Caenorhabditis elegans). Shading indicates residues that are identical (black) or conserved (grey) in five or more of the nine sequences, and with grey bars being predicted transmembrane domains. Vertebrates have two paralogues of Kish called TMEM167A and TMEM167B. (B) Growth of S. cerevisiae with a deletion in YNL024c-a (KSH1), the orthologue of CG14199. Strains were created that lacked the genomic copy of YNL024c-a but were kept alive by the YNL024c-a gene on a URA3 CEN ‘covering' plasmid, and also contained the indicated CEN (pRS415) or 2 μ LEU2 plasmids. The strains were streaked on 5-fluororotic acid (5-FOA) to eject the URA3 covering plasmid and then incubated for 3 days at 25°C before photography. The LEU2 plasmids containing YNL024c-a and TVP23 expressed from their own promoters support growth in the absence of genomic YNL024c-a, but this was not seen with the control plasmids (an empty CEN plasmid, or a 2μ plasmid expressing an irrelevant gene (LRC6)). (C) Wide-field micrographs of wild-type strain BY4741 with GFP integrated at the N or C terminus of the endogenous YNL024c-a gene by PCR-based homologous recombination. For the N-terminal fusion expression was promoted by a constitutive form of the PHO5 promoter. (D) Confocal micrographs of COS cells expressing the human orthologues of Kish with GFP attached to the C terminus, and labelled for the Golgi marker GM130.