Figure 5.

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.