A bipartite trigger for dislocation directs the proteasomal degradation of an endoplasmic reticulum membrane glycoprotein

Abstract

Polypeptides are organized into distinct substructures, termed protein domains, that are often associated with diverse functions. These modular units can act as binding sites, areas of post-translational modification, and sites of complex multimerization. The human cytomegalovirus US2 gene product is organized into discrete domains that together catalyze the proteasome-dependent degradation of class I major histocompatibility complex heavy chains. US2 co-opts the endogenous ER quality control pathway in order to dispose of class I. The US2 endoplasmic reticulum (ER)-lumenal region is the class I binding domain, whereas the carboxyl terminus can be referred to as the degradation domain. In the present study, we examined the role of the US2 transmembrane domain in virus-mediated class I degradation. Replacement of the US2 transmembrane domain with that of the CD4 glycoprotein completely blocked the ability of US2 to induce class I destruction. A more precise mutagenesis revealed that subregions of the US2 transmembrane domain differ in their ability to trigger class I degradation. Collectively, the data support a model in which US2-mediated class I degradation occurs as a highly regulated process where the US2 transmembrane domain and cytoplasmic tail work in concert to eliminate class I molecules. Host factors, including a signal peptidase complex, probably associate with the US2 molecule in a coordinated fashion to create a predislocation complex to promote the extraction of class I out of the ER. The results imply that the ER quality control machinery may recognize and eliminate misfolded proteins using a similar multistep regulated process.

Figure 1. Expression and characterization of HCMV US2 transmembrane domain chimeric constructs

A. Wild type US2₁₉₉ is a type I membrane protein consisting of an ER-lumenal domain (aa. 1-160) with a single glycosylation site (N68) (solid circles), a transmembrane domain (black box) (aa. 161-185), and a short cytoplasmic tail (solid line) (aa. 186-199) of 14 residues. The US2_CD4 chimera consists of the CD4 transmembrane domain (hatched box) and cytoplasmic tail (dashed line) (aa. 395-458) in place of the respective US2 sequences. The US2_CD4US2 chimera consists of the US2 lumenal domain, the CD4 transmembrane domain (aa. 395-419), and the US2 cytoplasmic tail. B. Total cell lysates (lanes 5-8 and 13-16) and US2 precipitates from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells using an anti-US2 serum (lanes 1-4 and 9-12) were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-US2 serum (lanes 1-8) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (lanes 9-16). C. US2 precipitates from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells using anti-US2 serum were digested with or without endoglycosidase H (1 hour, 37°C) (EndoH). Samples were resolved on SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-US2 serum. D. U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells plated onto coverslips were treated with anti-US2 polyclonal serum and anti-BiP monoclonal antibody, followed by incubation with a goat-anti-rabbit immunoglobulin^Fluorescein and a goat-anti-mouse immunoglobulin^{Texas Red}. A merged image of both stains is seen in the right hand column. For B and C, US2 proteins, GAPDH, and molecular standards are indicated.

Figure 1. Expression and characterization of HCMV US2 transmembrane domain chimeric constructs

A. Wild type US2₁₉₉ is a type I membrane protein consisting of an ER-lumenal domain (aa. 1-160) with a single glycosylation site (N68) (solid circles), a transmembrane domain (black box) (aa. 161-185), and a short cytoplasmic tail (solid line) (aa. 186-199) of 14 residues. The US2_CD4 chimera consists of the CD4 transmembrane domain (hatched box) and cytoplasmic tail (dashed line) (aa. 395-458) in place of the respective US2 sequences. The US2_CD4US2 chimera consists of the US2 lumenal domain, the CD4 transmembrane domain (aa. 395-419), and the US2 cytoplasmic tail. B. Total cell lysates (lanes 5-8 and 13-16) and US2 precipitates from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells using an anti-US2 serum (lanes 1-4 and 9-12) were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-US2 serum (lanes 1-8) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (lanes 9-16). C. US2 precipitates from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells using anti-US2 serum were digested with or without endoglycosidase H (1 hour, 37°C) (EndoH). Samples were resolved on SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-US2 serum. D. U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells plated onto coverslips were treated with anti-US2 polyclonal serum and anti-BiP monoclonal antibody, followed by incubation with a goat-anti-rabbit immunoglobulin^Fluorescein and a goat-anti-mouse immunoglobulin^{Texas Red}. A merged image of both stains is seen in the right hand column. For B and C, US2 proteins, GAPDH, and molecular standards are indicated.

Figure 2. US2 transmembrane chimeras fail to promote degradation of class I MHC molecules in a proteasomal-dependent fashion

Total cell lysates from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells untreated or treated with the proteasome inhibitor ZL₃VS (16 hrs, 2.5μM) were resolved on an SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-class I heavy chain serum (lanes 1-8) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (lanes 9-16). Glycosylated class I heavy chains (HC(+)CHO), deglycosylated class I heavy chains (HC(-)CHO), GAPDH, and molecular weight standards are indicated. The asterisk (*) represents nonspecific polypeptides.

Figure 3. US2 transmembrane domain chimeras fail to reduce the surface expression of class I MHC molecules

U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells were analyzed by flow cytometry. U373 (thick solid line) and US2-expressing cells (dashed lines) were labeled with W6/32 (A) or anti-HLA-A2 antibody (B) followed by an anti-mouse immunoglobulin conjugated to Alexa 647. In both experiments, U373 cells labeled with an immunoglobulin isotype control (Ig control, thin solid line) were included. The Alexa 647 fluorescence of surface class I molecules is represented as normalized cell number versus fluorescence signal.

Figure 4. US2 transmembrane domain chimeras continue to associate with class I MHC molecules

Top panel. Properly folded class I molecules were recovered from U373, US2₁₉₉, US2_CD4, and US2_CD4US2 cells with W6/32 (lanes 1-4). Bottom panel. US2 proteins were immunoprecipitated from the same samples with anti-US2 serum (lanes 5-8) to demonstrate equivalent amounts of US2 proteins. The precipitates were resolved on SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-class I heavy chain and anti-US2 sera. Class I heavy chains, US2 proteins, and molecular standards are noted.

Figure 5. Small adjustments to the US2_CD4US2 transmembrane domain do not restore US2 degradation abilities

A. Wild type US2₁₉₉, US2_CD4US2 (US2/CD4²⁵/US2), and transmembrane domain amino acid mutants US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 are depicted in a schematic diagram. The single letter amino acid code is used to identify the respective residue. The total number of residues in the transmembrane domain is indicated for each mutant. B. Properly folded class I molecules recovered from U373, US2₁₉₉, US2/CD4²⁵/US2, US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 cells with W6/32 were resolved on an SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-class I heavy chain serum. C. Total cell lysates from U373, US2₁₉₉, US2/CD4²⁵/US2, US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 cells were resolved on SDS-polyacrylamide gel (15%) and immunoblotted using anti-US2 serum (lanes 1-7) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (lanes 8-14). For B and C, class I heavy chains, US2 proteins, GAPDH, and molecular standards are indicated.

Figure 5. Small adjustments to the US2_CD4US2 transmembrane domain do not restore US2 degradation abilities

A. Wild type US2₁₉₉, US2_CD4US2 (US2/CD4²⁵/US2), and transmembrane domain amino acid mutants US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 are depicted in a schematic diagram. The single letter amino acid code is used to identify the respective residue. The total number of residues in the transmembrane domain is indicated for each mutant. B. Properly folded class I molecules recovered from U373, US2₁₉₉, US2/CD4²⁵/US2, US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 cells with W6/32 were resolved on an SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-class I heavy chain serum. C. Total cell lysates from U373, US2₁₉₉, US2/CD4²⁵/US2, US2/CD4²⁴/US2, US2/CD4²³/US2, US2/CD4²⁶/US2, and US2/CD4²⁷/US2 cells were resolved on SDS-polyacrylamide gel (15%) and immunoblotted using anti-US2 serum (lanes 1-7) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (lanes 8-14). For B and C, class I heavy chains, US2 proteins, GAPDH, and molecular standards are indicated.

Figure 6. Distinct regions within the US2 transmembrane domain are critical to mediating class I destruction

A. Wild type US2₁₉₉ and US2 transmembrane domain mutants 1 through 7 (US2_CD4US2 – 1 through US2_CD4US2 – 7) are depicted in a schematic diagram. Sequences from the US2 transmembrane domain are shown within black lines; sequences from the CD4 transmembrane domain are shown within gray lines. The single letter amino acid code is used to identify the respective residue. B. Total cell lysates of U373, US2₁₉₉, and US2 transmembrane domain mutants 1 through 7 were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-class I heavy chain serum (lanes 1-9). The asterisk (*) represents nonspecific polypeptides. C. US2 proteins recovered from wild type US2₁₉₉ and US2 transmembrane domain mutants 1 through 7 using anti-US2 were resolved on SDS-PAGE (15%) and subjected to an anti-US2 immunoblot (lanes 1-9). For B and C, class I heavy chains, US2 proteins, and molecular standards are indicated.

Figure 6. Distinct regions within the US2 transmembrane domain are critical to mediating class I destruction

A. Wild type US2₁₉₉ and US2 transmembrane domain mutants 1 through 7 (US2_CD4US2 – 1 through US2_CD4US2 – 7) are depicted in a schematic diagram. Sequences from the US2 transmembrane domain are shown within black lines; sequences from the CD4 transmembrane domain are shown within gray lines. The single letter amino acid code is used to identify the respective residue. B. Total cell lysates of U373, US2₁₉₉, and US2 transmembrane domain mutants 1 through 7 were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-class I heavy chain serum (lanes 1-9). The asterisk (*) represents nonspecific polypeptides. C. US2 proteins recovered from wild type US2₁₉₉ and US2 transmembrane domain mutants 1 through 7 using anti-US2 were resolved on SDS-PAGE (15%) and subjected to an anti-US2 immunoblot (lanes 1-9). For B and C, class I heavy chains, US2 proteins, and molecular standards are indicated.

Figure 7. US2_CD4US2 transmembrane domain mutants 1 through 7 continue to associate with class I molecules

Properly folded class I molecules were recovered using W6/32 from U373 cells, US2₁₉₉ cells, and cells expressing US2 transmembrane domain mutants 1 through 7 (US2_CD4US2 – 1 through US2_CD4US2 – 7). The cells were treated with the proteasome inhibitor ZL₃VS (2.5μM, 16 hrs). Precipitates were resolved on an SDS-polyacrylamide gel (15%) and subjected to immunoblot analysis using anti-class I heavy chain (lanes 1-9) and anti-US2 sera (lanes 10-18). Class I heavy chains, US2 proteins, and molecular standards are noted. The asterisk (*) represents nonspecific polypeptides.

Figure 8. US2_CD4US2 transmembrane domain mutants 1 through 7 complex with signal peptide peptidase

U373, US2₁₉₉, and mutants US2_CD4US2 – 1 through US2_CD4US2 – 7 cells treated with the proteasome inhibitor ZL₃VS (2.5μM, 16 hrs) were subjected to immunoprecipitation using anti-signal peptide peptidase sera. Samples were resolved on SDS-polyacrylamide gel (12.5%) and subjected to immunoblot analysis using anti-US2 (lanes 1-9) and anti-signal peptide peptidase (SPP) (lanes 10-18) sera. US2 proteins, SPP, immunoglobulin heavy chain (Ig HC), and molecular standards are indicated. The asterisk (*) represents slower migrating US2 polypeptides.

Figure 9. Proposed models of US2-mediated dislocation of class I MHC molecules

HCMV US2 induces the degradation of class I heavy chains in a regulated, multi-step process that probably involves various cellular components. The initial step toward class I dislocation may transpire in one of two manners: (1) US2/class I interaction via its ER-lumenal domain would result in the recruitment of cellular factors X, Y, and Z to the transmembrane domain followed by the engagement of signal peptide peptidase (SPP) or (2) US2 would interact with SPP and possibly factors X, Y, and Z through its carboxy-terminus prior to binding to class I molecules. Once this pre-dislocation complex of US2, class I, and cellular factors is assembled, (3) the dislocation message is relayed to the US2 cytoplasmic tail probably through the transmembrane domain. The US2 tail would orient itself into a 3₁₀-helix. This would allow the four critical residues (cysteine¹⁸⁷, serine¹⁹⁰, tryptophan¹⁹³, and phenylalanine¹⁹⁶) of the cytoplasmic tail to make contact with the cellular dislocation machinery. (4) Class I heavy chains would then rapidly extracted from the ER membrane through the dislocation pore and (5) deposited into the cytosol where they would be destroyed by the proteasome.