Misfolded major histocompatibility complex class I heavy chains are translocated into the cytoplasm and degraded by the proteasome

Abstract

N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL), which reversibly inhibits the proteasome in addition to other proteases, and a more specific irreversible inhibitor of the proteasome, lactacystin, were found to cause the accumulation of major histocompatibility complex (MHC) class I heavy chains in the cytosol of the beta2-microglobulin-deficient cell line Daudi and the TAP-deficient cell line .174. These cell lines, which are severely impaired in their ability to fold MHC class I heavy chain, showed an accumulation of soluble class I heavy chains at different rates over a period of hours in the presence of LLnL. The accumulation of soluble class I heavy chains in the presence of either LLnL or lactacystin was easily revealed in Daudi and .174 but almost undetectable in a Daudi transfectant expressing beta2-microglobulin and in 45.1, the wild-type parent of .174. The soluble class I heavy chain was also found to be devoid of its N-linked glycan and to be located in the cytosol. When the gene for ICP47, a herpes simplex virus protein that blocks the translocation of peptides into the endoplasmic reticulum, was transfected into 45.1, a similar accumulation of soluble MHC class I heavy chain was detectable. These data suggest that in cells where the MHC class I molecule is unable to assemble properly, the misfolded heavy chain is removed from the endoplasmic reticulum to the cytosol, deglycosylated, and degraded by the proteasome.

Figure 1

Proteasome inhibitors induce the accumulation of a MHC class I heavy chain derivative in Daudi and .174 cells but not in their wild-type counterparts. Daudi, Daudi.β₂m, .174, and 45.1 cells were incubated in 250 μM LLnL, 20 μM lactacystin, or DMSO solvent control for 2 hr at 37°C and separated into membrane (M) and soluble (S) fractions by ultracentrifugation. MHC class I heavy chain was immunoprecipitated after addition of Triton X-100 using mouse antibody HC10, subjected to SDS/PAGE, transferred to Immobilon P membrane, and visualized using the rat antibody 3B10.7.

Figure 2

Kinetic analysis of the accumulation of the MHC class I heavy chain derivative in Daudi (A) and .174 (B). Cells were preincubated for 1 hr with 250 μM LLnL or DMSO solvent control at 37°C, pulsed for 10 min with 2.0 mCi of [³⁵S]methionine, and chased for 6 hr. At the indicated time points, samples were separated into membrane and soluble fractions by ultracentrifugation. MHC class I heavy chain was precipitated using the mouse antibody HC10 and then re-precipitated using the rat antibody 3B10.7 and subjected to SDS/PAGE. One-eighth of the membrane fraction from the zero time point was treated in the presence or absence of N-glyF and loaded to serve as a reference for deglycosylated class I heavy chain. Filled arrowheads indicate glycosylated class I heavy chain. Shaded arrowheads indicate deglycosylated class I heavy chain.

Figure 3

The LLnL-induced MHC class I heavy chain derivative is not glycosylated. Daudi (A) and .174 (B) cells were treated with 250 μM LLnL or DMSO solvent control for 2 hr at 37°C, separated into membrane and soluble fractions, immunoprecipitated using the mouse antibody HC10, treated with endo H or N-glyF, subjected to SDS/PAGE, transferred to Immobilin P membrane, and visualized using the rat antibody 3B10.7. Solid arrowheads indicate glycosylated class I heavy chain. Shaded arrowheads indicate deglycosylated class I heavy chain.

Figure 4

The LLnL-induced MHC class I heavy chain derivative is located in the cytosol. Daudi and .174 cells were treated with 250 μM LLnL or DMSO solvent control for 4 hr at 37°C. The cells were treated with SLO for 30 min at 20°C and separated into lumenal (L) or cytosolic (C) fractions by centrifugation. Class I heavy chains were immunoprecipitated using HC10, subjected to SDS/PAGE, and visualized using 3B10.7. The soluble, ER-retained chaperone, grp94 (gp96), was immunoprecipitated and visualized using the rat mAb clone 9G10. The soluble, cytosolic control molecule, Hsc70, was immunoprecipitated and visualized using the rat antibody 1B5.

Figure 5

ICP47 induces the soluble MHC class I heavy chain derivative in 45.1. (A) The cells lines 45.1 and 45.1.ICP47 were analyzed for class I surface expression by flow cytometry using the mAb w6/32 and fluorescein-conjugated rabbit anti-mouse IgG. The control was a nonspecific isotype-matched mAb. (B) The lines 45.1 and 45.1.ICP47 were treated with 250 μM LLnL for 2 hr at 37°C and separated into membrane (M) and soluble (S) fractions by ultracentrifugation. MHC class I heavy chain was immunoprecipitated after addition of Triton X-100 using mouse antibody HC10, subjected to SDS/PAGE, transferred to Immobilon P membrane, and visualized using the rat antibody 3B10.7.