US2-mediated breakdown of Class I MHC HC can be disrupted by attenuation of β₂m expression levels. (A) Control cells (control; lanes 1–3), US2-expressing cells (control+US2; lanes 4–6), β₂m siRNA cells (β₂m^RNAi; lanes 7–9) and β₂m siRNA plus US2-expressing cells (β₂m^RNAi+US2; lanes 10–12) were pulse-labeled with ³⁵S-methionine for 10 min and chased for the indicated times. Cells were lysed in NP-40 lysis buffer, followed by immunoprecipitation for properly folded Class I MHC (W6/32; panel (A)), Class I MHC HC (αHC; panel (B)), US2 (αUS2; panel (C)) and the TfR (panel (D)). Indicated on the left-hand side are the migration of the glycosylated (+CHO) and deglycosylated (−CHO) form of Class I MHC HC, respectively, or glycosylated and deglycosylated US2 (panel C). TfR_HM and TfR_C indicate the migration of the TfR containing high-mannose- and complex-type sugars, respectively (panel (D)). (E) Cell extracts of US2-expressing cells (control+US2; lanes 1–6) and β₂m siRNA plus US2-expressing cells (β₂m^RNAi+US2; lanes 7–12) were labeled as described in (A) and subjected to subcellular fractionation by ultracentrifugation at 100 000 g (described in Tortorella et al, 1998). The obtained fractions were subsequently used to recover Class I MHC HC by immunoprecipitation with the HC70 antibody. The migration of the glycosylated (+CHO) and deglycosylated (−CHO) form of Class I MHC HC is indicated on the left-hand side.

PNGase is responsible for deglycosylation of Class I MHC HCs in US2-mediated dislocation. (A) US2-expressing cells (control+US2; lanes 1–3) and US2-expressing PNGase siRNA cells (PNG1^RNAi+US2; lanes 4–6) were pulse-labeled with ³⁵S-methionine for 10 min and chased for the indicated times. NP-40 cell extracts were used for the immunoprecipitation of correctly folded Class I MHC (W6/32). To demonstrate downregulation of Class I MHC molecules, control cells were run in parallel (control; left-hand side). (B) Same as in (A); NP-40 cell extracts were used for the immunoprecipitation of free Class I MHC HC (αHC). The migration of glycosylated (+CHO) and deglycosylated (−CHO) HCs, respectively, is indicated on the left-hand side. (C) Same as in (A); NP-40 extracts were used for the immunoprecipitation of US2 (αUS2), which migrates as a doublet. (D) Same as in (A); NP-40 cell extracts were used for the immunoprecipitation of the TfR glycoprotein. Slower migration of the TfR at later chase points is due to the processing of high-mannose N-linked sugars.

Quantitation of dislocated Class I MHC HC in US2- and US2 plus PNG^RNAi-expressing cells. (A) Radioactive signal of glycosylated (+CHO) and deglycosylated (−CHO) HC shown in Figure 5 was quantitated by use of a Phosphorimager for both US2- and US2 plus PNG1^RNAi-expressing cells. Signals were expressed as the percent HC recovered at the indicated time points relative to the 0-min time point of each cell line. The sum of the amount of glycosylated HC and deglycosylated HC in the 100 000 g pellet fraction at 0, 20 and 40 min is depicted. White bars represent the total amount of Class I HC in US2-expressing cells, and light gray bars represent that in US2 plus PNG1^RNAi-expressing cells. (AU=arbitrary units). (B) Depiction of the ratio of glycosylated versus deglycosylated HCs in the 100 000 g supernatant fraction in US2-expressing cells (black bars) and US2 plus PNG1^RNAi-expressing cells (white bars).

Attenuation of β₂m expression levels in U373 astrocytoma cells. (A) Western blot analyses of U373 control cells and β₂m siRNA cells. Total extracts (30 μg per lane) of U373 control cells (wt) and U373 cells infected with two independent β₂m siRNA constructs (I and II, respectively) were separated by SDS–PAGE, blotted onto PVDF and tested for the presence of β₂m. (B) Same as in (A); extracts were tested for the presence of Class I MHC HC. (C) Same as in (A); extracts were tested for the presence of PDI. Note that extracts from two independent cell lines harboring the β₂m siRNA construct II were tested, since the complete nucleotide sequence of this construct could not be determined.

PNGase siRNA cells display normal glycoprotein turnover. (A) PNGase siRNA cell lines were generated as described in Materials and methods. NP-40 cell extracts (575 μg) were tested for PNGase activity in a deglycosylation assay using radiolabeled TCRα as a substrate (Hirsch et al, 2003). Lane 1: TCRα incubated without cell extracts (−). Lane 2: TCRα incubated with NP-40 extracts from U373 control cells (wt). Lanes 3–7: TCRα incubated with NP-40 extracts from cells infected with the indicated PNGase siRNA constructs (I–IV). Note that extracts from two independent cell lines harboring the PNGase siRNA construct I were tested, since the complete nucleotide sequence of this construct could not be determined. The number of N-linked glycans of TCRα is indicated on the right-hand side. Note that the doublet observed for TCRα carrying two residual N-linked glycans is most probably due to heterogeneous deglycosylation of the polypeptide, since the four N-linked attachment sites of TCRα allow six different glycosylation patterns. (B) Control cells (control; lanes 1–3) and PNGase siRNA cells (PNG1^RNAi; lanes 4–6) were pulse-labeled with ³⁵S-methionine for 10 min and chased for the indicated times. Cells were lysed in NP-40 lysis buffer followed by immunoprecipitation for properly folded Class I MHC (W6/32; upper panel) or Class I MHC HC (αHC; lower panel). (C) Same procedure as in (B); NP-40 cell extracts were used for immunoprecipitation of the TfR (α-TfR).

Glycosylated Class I MHC HCs accumulate in the cytosol after dislocation in PNGase knockdown cells. (A) US2-expressing cells (control+US2; lanes 1–3, 7–9 and 13–15) and US2-expressing PNGase siRNA cells (PNG1^RNAi+US2; lanes 4–6, 10–12 and 16–18) were pulse-labeled with ³⁵S-methionine for 10 min and chased for 0, 20 and 40 min. Cells were resuspended in 250 mM sucrose containing 10 mM Tris, pH 7.4 and 50 mM NaCl and passed through a ball bearing homogenizer 14 times. One quarter of this homogenate was used to immunoprecipitate Class I MHC HC (total; lanes 1–3 and 4–6). The remaining three quarters were spun at 100 000 g for 1 h. The supernatant fraction was then adjusted to 0.5% NP-40 and 125 mM sucrose; the pellet fraction was resuspended in NP-40 lysis buffer. Both fractions were used for the immunoprecipitation of Class I MHC HC. (B) Same as in (A), fractions were used for the immunoprecipitation of the ER resident protein calnexin. (C) Same as in (A), fractions were used for the immunoprecipitation of fully assembled Class I MHC by use of the W6/32 monoclonal antibody. Note that part (C) was exposed 10 times longer than part (A), in order to detect W6/32-reactive Class I MHC, which is downregulated in US2-expressing cells. Note that lanes 1–6 contain one-third of the amount used in lanes 7–18 for each immunoprecipitation.