Journal: Nature

Article Title: An intramembrane chaperone complex facilitates membrane protein biogenesis

doi: 10.1038/s41586-020-2624-y

Figure Lengend Snippet: Stable cell lines containing the indicated inducible reporters were treated with scrambled or Asterix-targeting siRNAs, reporter expression was induced for ∼6 hours, and the cells were analysed by flow cytometry. A cartoon depicting the topology, number of TMDs, and fluorescent proteins for each of the membrane protein reporters is show to the left of its respective flow cytometry data. The viral P2A peptide sequence results in two proteins from a single translation reaction as indicated. The plots show histograms of fluorescent protein ratios in control cells (grey) and Asterix-knockdown cells (blue). The dashed black line indicates the mode for the control population.

Article Snippet: Cells were passed through a 70μm filter before flow cytometry analysis using a Beckton Dickinson LSRII instrument.

Techniques: Stable Transfection, Expressing, Flow Cytometry, Membrane, Sequencing, Control, Knockdown

Journal: Nature

Article Title: An intramembrane chaperone complex facilitates membrane protein biogenesis

doi: 10.1038/s41586-020-2624-y

Figure Lengend Snippet: (a) The raw data for three of the histograms of the GFP:RFP ratio (or RFP:GFP ratio in the case of GFP-2A-RFP-SQS) shown in Fig. 3. The mode of the control histogram (dotted black line in Fig. 3) was used to determine the statistical mode of GFP:RFP (or RFP:GFP) ratio. This mode was used as a gate to colour the dot plots shown below the histograms such that all cells above the gate were coloured red and those below the gate were coloured grey. The percent of cells above the gate for each plot is indicated. (b) Flow- cytometry analysis of the indicated GPCR reporters using the dual-colour assay system exactly as in Fig. 3. Cell lines containing the reporter stably integrated at a single FRT site located downstream of a doxycycline-inducible reporter were used for these assays. This allows assay of cells using a single transfection (which proved to be less toxic than sequential transfections with both the siRNA and the reporter), and provided control of the length of time of reporter expression. Each reporter cell line was treated with scrambled (Scr) or Asterix-targeting siRNAs, then at the time of effective knockdown (verified in separate experiments using immunoblotting), the reporter was induced for ∼6-8 hours. Induction only after knockdown allows us to monitor the reporter that was produced in the absence of Asterix rather than a heterogeneous mixture of reporter expressed during the knockdown. The histograms of the GFP:RFP ratio in the scrambled- versus Asterix-siRNA cells are shown in grey and blue, respectively, in the upper plot for each construct. The two dot plots below the histogram are the corresponding raw data plotted as described in panel a. Each reporter shows a distribution of lower GFP:RFP ratio, with some reporters being more impacted than others. This is not seen with the tail-anchored protein SQS using the same assay format.

Article Snippet: Cells were passed through a 70μm filter before flow cytometry analysis using a Beckton Dickinson LSRII instrument.

Techniques: Control, Flow Cytometry, Stable Transfection, Transfection, Expressing, Knockdown, Western Blot, Produced, Construct

Journal: Nature

Article Title: An intramembrane chaperone complex facilitates membrane protein biogenesis

doi: 10.1038/s41586-020-2624-y

Figure Lengend Snippet: (a) The diagrams depict dual-colour fluorescent reporters for protein stability as an indirect measure of successful biogenesis. The membrane protein of interest is tagged with one fluorescent protein (FP), which is separated from a second FP by the viral 2A peptide sequence. When the 2A sequence is translated, peptide bond formation is skipped without perturbing elongation by the ribosome. Thus, translation results in two separate proteins made in a 1:1 stoichiometry that are separated at the 2A sequence. If biogenesis of the membrane protein is impaired, it will be degraded along with its tagged FP, resulting in an altered ratio of the two FPs. Thus, treatment conditions that impair biogenesis of the membrane protein will be reflected as a relative change in the ratio of FPs. The three reporters encoding angiotensin type-2 receptor II (AGTR2), squalene synthase (SQS) and Asialglycoprotein receptor (ASGR) were transiently transfected into wild-type (WT), CCDC47 KO (ΔCCDC47) or Asterix KO (AAsterix) HEK293 cells and analysed by dual- colour flow cytometry. Histograms represent the distribution of FP ratio in WT (grey), ΔCCDC47 (red) and AAsterix (blue) cells. A biogenesis defect is only seen for the multi- spanning membrane protein AGTR2, but not for the tail-anchored protein SQS or the signal- anchored single pass protein ASGR. (b) Assays similar to those in Fig. 3, but for cell lines treated with scrambled versus CCDC47 siRNAs as indicated. We find that the phenotypes for Asterix and CCDC47 knockdowns are very similar for all reporters (three are shown here), with CCDC47 consistently being somewhat more modest. The reason for this seems to be that CCDC47 knockdown is slower and less efficient than Asterix knockdown. Note that similar phenotypes are seen for AGTR2 and SS-T4L-AGTR2, a version that contains an N- terminal signal sequence and T4 lysozyme preceding TMD1. In earlier studies, we found that initiating translocation with a signal sequence completely bypasses the requirement for EMC- mediated TMD1 insertion. The fact that SS-T4L-AGTR2 remains sensitive to PAT complex depletion (as judged by either Asterix or CCDC47 knockdowns) argues that the PAT complex acts independently of EMC.

Article Snippet: Cells were passed through a 70μm filter before flow cytometry analysis using a Beckton Dickinson LSRII instrument.

Techniques: Membrane, Sequencing, Transfection, Flow Cytometry, Knockdown, Translocation Assay