Volume 23, Issue 10, Pages 3068-3077 (June 2018) Cytomegalovirus gp40/m152 Uses TMED10 as ER Anchor to Retain MHC Class I  Venkat Raman Ramnarayan, Zeynep Hein, Linda Janßen, Natalia Lis, Swapnil Ghanwat, Sebastian Springer  Cell Reports  Volume 23, Issue 10, Pages 3068-3077 (June 2018) DOI: 10.1016/j.celrep.2018.05.017 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions

Figure 1 The gp40 Linker Controls gp40 Trafficking and MHC Class I Retention (A) Schematic representation of wild-type gp40 and linker quarter mutants. Indicated are the folded lumenal domain, the linker, the transmembrane domain (TM) and cytoplasmic tail (CY), the hemagglutinin (HA) tag at the C terminus, and the residue numbers of the mature protein. The wild-type linker consists of four quarters (L1–L4), which were individually (GLLL, LGLL, LLGL, and LLLG) or collectively (GGGG) replaced with (Gly4Ser)2 sequences. The previously described gp40-(G4S)9 mutant is thus represented here as GGGG (Figure 5A in Janßen et al., 2016). (B) The first three quarters of the linker are necessary for class I retention. gp40 linker mutant-expressing murine fibroblasts were surface-stained for flow cytometry with B22.249 (Db) and Y3 (Kb). The mean fluorescence intensity of cell surface class I was analyzed by flow cytometry and is represented in a bar chart (mean ± SEM, n = 4). (C) The class I-retaining forms of gp40 cycle in the early secretory pathway. HEK293T cells expressing wild-type and mutant gp40 forms were pulse-labeled for 10 min and chased for the indicated times and lysed in 1% Triton X-100 buffer. gp40 was immunoprecipitated with anti-HA antibody, digested with EndoF1 or PNGase as indicated, and separated by SDS-PAGE. Rt, sialylated band; S, EndoF1-sensitive band; black arrows, partial-EndoF1-resistant band; asterisk, unspecific band. One representative experiment out of two is shown. Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions

Figure 2 The p24 Family Member TMED10 Binds to gp40 in a Linker-Dependent but Class I-Independent Manner (A) p24 proteins co-precipitate with gp40. Murine fibroblasts expressing gp40-LLLL, empty vector, or gp40-GGGG were lysed in 1% digitonin buffer. gp40 was immunoprecipitated with anti-HA antibody, separated on a 10%–12.5% gradient gel by SDS-PAGE, and silver-stained. Bands indicated by black arrows were excised and sequenced. The table indicates the corresponding proteins and their abundance. One representative experiment out of two is shown. (B) The slow-trafficking forms of gp40 bind TMED10. Murine fibroblasts expressing the gp40 linker quarter mutants were lysed in 1% digitonin buffer. gp40 was immunoprecipitated using anti-HA antibody, and the proteins were separated by SDS-PAGE and immunoblotted against gp40 and TMED10. (C) Linker-mediated gp40 retention is class I independent. Class I-deficient murine melanoma cells, without (left) or with (right) re-introduced class I (Db and Kb), and expressing gp40-LLLL or gp40-GGGG, were pulse-labeled for 10 min and chased for the indicated times and then lysed in 1% Triton X-100 buffer. gp40 was immunoprecipitated with anti-HA antibody, digested with EndoF1 as indicated, and separated by SDS-PAGE. Rt, sialylated band; S, EndoF1-sensitive band. One representative experiment out of two is shown. (D) TMED10 binding of gp40 is class I independent. Class I-deficient murine melanoma cells, without (right) or with (left) re-introduced class I (Db and Kb), and expressing gp40-LLLL were lysed in 1% digitonin buffer. gp40 was immunoprecipitated using anti-HA antibody, and the proteins were separated by SDS-PAGE and immunoblotted against gp40 and TMED10. Class I was immunoprecipitated using anti-Kb/Db antiserum and immunoblotted using the same antibody. (E) gp40 tethers class I to TMED10. K41 cells expressing gp40-LLLL, gp40-GGGG, or an empty vector were lysed in 1% digitonin buffer. Class I was immunoprecipitated using Db/Kb serum against the common sequence in cytosolic tail, and the proteins were separated by SDS-PAGE and immunoblotted against gp40 and TMED10. Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions

Figure 3 gp40 Requires the p24 Proteins for MHC Class I Retention (A) Knockdown of p24 proteins influences TMED10 levels differently. Murine fibroblasts were transfected with the indicated siRNAs and lysed in 1% Triton X-100 buffer, and the proteins were separated using SDS-PAGE and immunoblotted against calnexin (top) and TMED10 (bottom). Also see Figure S5. (B) Loss of p24 proteins restores cell surface class I levels. Murine fibroblasts expressing either the empty vector (-gp40) or gp40 were transfected with 300 pmol of the indicated siRNA. After 60 hr, the cells were harvested and co-stained for cell surface class I and CD29. The cells were analyzed by flow cytometry, and two-dimensional plots of cell surface class I levels (x axis) and CD29 (y axis) are depicted here (also see Figure S4). The dotted line within each panel bifurcates cells with either high or low cell surface class I levels, and the percentage of cells thereof are presented within the panels. One representative experiment out of six is shown. (C) The percentage values shown in (B) are quantified as mean ± SEM of six independent experiments and normalized to Tmed10 siRNA knockdown. (D) Murine fibroblasts previously expressing gp40-LLLL and gp40-GGGG were transduced with anti-TMED10 shRNA vectors. The cells were stained with B22.249 (H-2Db) or Y3 (H-2Kb) and analyzed using flow cytometry. Showing the mean fluorescence intensities normalized to cells expressing the retention incompetent gp40-GGGG mutant. Mean ± SEM of two independent experiments. (E) Loss of TMED10 destabilizes gp40-LLLL but not gp40-GGGG. Cells in (D) were pulse-labeled for 10 min and chased for the indicated times and lysed with 1% Triton X-100 buffer. gp40 was immunoprecipitated with anti-HA antibody, digested with EndoF1 as indicated, and separated by SDS-PAGE. Rt, sialylated band; S, EndoF1-sensitive band. (F) In the absence of TMED10, intracellular gp40 is degraded, cell surface class I is restored. gp40-expressing NIH 3T3 cells were transfected with siRNA against Tmed10 (no siRNA for control cells), fixed, and permeabilized. Next, the cells were stained with anti-class I antibody 27.11.13S and Alexa Fluor 488-conjugated secondary, gp40 with Alexa Fluor 647-conjugated anti-HA antibody. KD, knockdown cells; WT, wild-type. Scale bar: 20 μm. Also see Figures S8 and S9. Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions

Figure 4 The Trafficking Motifs of TMED10 Regulate the Trafficking of gp40 and Class I Retention (A) Schematic representations of wild-type TMED10 and TMED10-AA/SS mutant. Indicated are the FLAG tag (FL) at the N terminus, the folded GOLD domain, the coiled-coil domain (CC), the transmembrane domain (TM) and cytoplasmic tail (CY), and the trafficking signals (or the respective mutations). On the right, general localization of the proteins within the cell is mentioned (Nickel et al., 1997). Also see Figure S6. (B) Overexpression of TMED10-AA/SS mutant restores cell surface class I levels. Mouse fibroblasts expressing gp40-LLLL-GFP were transduced either with wt-TMED10 or TMED10-AA/SS mutant. The cells were stained with B22.249 (H-2Db) or Y3 (H-2Kb) and analyzed using flow cytometry. One representative experiment out of two is shown. (C) The trafficking signals in TMED10 anchor gp40 to the ER. HEK293T cells were co-transfected with gp40-LLLL and either wt-TMED10 (top) or TMED10-AA/SS (bottom). The cells were pulse labeled for 10 min and chased for the indicated times, then lysed in 1% digitonin. The TMED10 forms were immunoprecipitated first with anti-FLAG antibody, and after denaturation in 1% SDS, gp40 was re-immunoprecipitated with anti-HA antibody. The proteins were digested with EndoF1 as indicated and separated by SDS-PAGE. Rt, sialylated band; S, EndoF1-sensitive band. Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Proposed Mechanism for gp40-Mediated Class I Retention via TMED10 (A) In the absence of gp40, class I heavy chains (dark red) bind to β2m (pink) and high-affinity peptide (blue) before being exported to the cell surface. Once they pass the medial Golgi, they become fully EndoF1 resistant. (B) In gp40-expressing cells, fully assembled and peptide-loaded class I molecules are bound in the ER by gp40 (orange), which itself is bound to TMED10 (green) via the gp40 linker, and through the trafficking signals of TMED10, the complex is returned to the ER. Cycling in the early secretory pathway leads to a slow maturation of the glycans of both gp40 and class I (Figure 1F in Janßen et al., 2016) and results in their partial EndoF1 resistance. (C) Knockdown (KD) of TMED10 in gp40-expressing cells destabilizes gp40 (blurred) in an unknown manner, allowing class I cell surface trafficking. (D) In cells overexpressing the TMED10-AA/SS mutant (green with modified cytosolic domain), binding of gp40 to TMED10-AA/SS causes the rapid exit of gp40 from the ER (black double arrow), thus depriving gp40 of binding class I. As a result, cell surface class I levels are restored. (E) gp40-GGGG (orange with a modified linker), which cannot bind TMED10, is rapidly exported out of the ER (black double arrows) and therefore does not bind to class I, which moves to the cell surface. Cell Reports 2018 23, 3068-3077DOI: (10.1016/j.celrep.2018.05.017) Copyright © 2018 The Authors Terms and Conditions