Dislocation of an Endoplasmic Reticulum Membrane Glycoprotein Involves the Formation of Partially Dislocated Ubiquitinated Polypeptides

Abstract

Accumulation of improperly folded polypeptides in the endoplasmic reticulum (ER) can trigger a stress response that leads to the export of aberrant proteins into the cytosol and their ultimate proteasomal degradation. Human cytomegalovirus encodes a type I glycoprotein, US11, that binds to nascent MHC class I heavy chain molecules and causes their dislocation from the ER to the cytosol where they are degraded by the proteasome. Examination of US11-mediated class I degradation has identified a host of cellular proteins involved in the dislocation reaction, including the cytosolic AAA ATPase p97, the membrane protein Derlin-1, and the E3 ubiquitin ligase Sel1L. However, the intermediate steps occurring between the initiation of dislocation and full extraction of the misfolded substrate into the cytosol are not known. We demonstrate that US11 itself undergoes ER export and proteasomal degradation and utilize this system to define multiple steps of US11 dislocation. Treatment of US11-expressing cells with proteasome inhibitor resulted in the accumulation of glycosylated and ubiquitinated species as well as a deglycosylated US11 intermediate. Subcellular fractionation of proteasome-inhibited US11 cells demonstrated that deglycosylated intermediates continued to be integrated within the ER membrane, suggesting that the proteasome functions in the latter steps of dislocation. The data supports a model in which US11 is modified with ubiquitin, whereas the transmembrane region is integrated in the ER membrane, and deglycosylation occurs before complete dislocation. Accumulation of improperly folded polypeptides in the endoplasmic reticulum (ER) can trigger a stress response that leads to the export of aberrant proteins into the cytosol and their ultimate proteasomal degradation. Human cytomegalovirus encodes a type I glycoprotein, US11, that binds to nascent MHC class I heavy chain molecules and causes their dislocation from the ER to the cytosol where they are degraded by the proteasome. Examination of US11-mediated class I degradation has identified a host of cellular proteins involved in the dislocation reaction, including the cytosolic AAA ATPase p97, the membrane protein Derlin-1, and the E3 ubiquitin ligase Sel1L. However, the intermediate steps occurring between the initiation of dislocation and full extraction of the misfolded substrate into the cytosol are not known. We demonstrate that US11 itself undergoes ER export and proteasomal degradation and utilize this system to define multiple steps of US11 dislocation. Treatment of US11-expressing cells with proteasome inhibitor resulted in the accumulation of glycosylated and ubiquitinated species as well as a deglycosylated US11 intermediate. Subcellular fractionation of proteasome-inhibited US11 cells demonstrated that deglycosylated intermediates continued to be integrated within the ER membrane, suggesting that the proteasome functions in the latter steps of dislocation. The data supports a model in which US11 is modified with ubiquitin, whereas the transmembrane region is integrated in the ER membrane, and deglycosylation occurs before complete dislocation. The endoplasmic reticulum (ER) 3The abbreviations used are:ERendoplasmic reticulumMHCmajor histocompatibility complexUSunique shortHCMVhuman cytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenaseTRAM1translocating chain-associated membrane protein-1ZL3VScarboxybenzyl-leucylleucyl-leucyl vinyl sulfoneIEFisoelectric focusingWTwild type 3The abbreviations used are:ERendoplasmic reticulumMHCmajor histocompatibility complexUSunique shortHCMVhuman cytomegalovirusGAPDHglyceraldehyde-3-phosphate dehydrogenaseTRAM1translocating chain-associated membrane protein-1ZL3VScarboxybenzyl-leucylleucyl-leucyl vinyl sulfoneIEFisoelectric focusingWTwild type of eukaryotic cells is the site of synthesis of membrane and secretory proteins. Polypeptides are cotranslationally inserted into the ER lumen where they can be modified by the addition of asparagine-linked glycan precursors. Proteins achieve proper tertiary structure with the aid of ER resident molecules such as calnexin, calreticulin, ERp57, and protein disulfide isomerase (1Jessop C. Chakravarthi S. Garbi N. Hammerling G. Lovell S. Bulleid N. EMBO J. 2007; 1: 28-40Crossref Scopus (161) Google Scholar, 2Creighton T. Hillson D. Freedman R. J. Mol. Biol. 1980; 142: 43-62Crossref PubMed Scopus (145) Google Scholar, 3Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (716) Google Scholar, 4Nigam S.K. Goldberg A.L. Siew Ho Rohde M.F. Bush K.K. Sherman M.Y. J. Biol. Chem. 1994; 269: 1744-1749Abstract Full Text PDF PubMed Google Scholar). Around 30% of newly synthesized proteins are unable to assemble into their final functional conformation or complex and are degraded (5Schubert U. Anton L. Gibbs J. Norbury C. Yewdell J.W. Bennick J. Nature. 2000; 404: 770-774Crossref PubMed Scopus (1) Google Scholar). Improperly assembled proteins are recognized by the ER quality control machinery and exported from the ER in a process called dislocation and are degraded by the proteasome. The ability of the cell to dispose of misfolded proteins is critical for cell survival (6Faitova J. Krekac D. Hrstka R. Vojtesek B. Cell. Mol. Biol. Lett. 2006; 11: 488-505Crossref PubMed Scopus (136) Google Scholar). The accumulation of misfolded proteins within the ER can result in the activation of an ER stress signaling pathway that aims to restore cellular homeostasis through transcriptional and translational alterations. This may result in up-regulation of ER folding chaperones and inhibition of eIF2α-dependent translation (7Rutkowski D. Kaufman R. Trends Cell Biol. 2004; 14: 20-28Abstract Full Text Full Text PDF PubMed Scopus (1184) Google Scholar, 8Harding H. Calfon M. Urano F. Novoa I. Ron D. Annu. Rev. Cell Dev. Biol. 2002; 18: 575-599Crossref PubMed Scopus (808) Google Scholar). endoplasmic reticulum major histocompatibility complex unique short human cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase translocating chain-associated membrane protein-1 carboxybenzyl-leucylleucyl-leucyl vinyl sulfone isoelectric focusing wild type endoplasmic reticulum major histocompatibility complex unique short human cytomegalovirus glyceraldehyde-3-phosphate dehydrogenase translocating chain-associated membrane protein-1 carboxybenzyl-leucylleucyl-leucyl vinyl sulfone isoelectric focusing wild type The removal and degradation of aberrant proteins in the ER is a multifaceted process that likely occurs through several steps. First, degradation substrates are recognized by ER sensors and targeted for removal via an aqueous membrane pore referred to as the “dislocon.” Ubiquitination of ER degradation substrates is carried out by cytosolic or ER membrane bound E3 ubiquitin ligases. Once exposed to the cytosolic environment, N-linked glycans can be cleaved by the cytosolic enzyme peptide N-glycanase. Finally, the ER degradation substrates are delivered to the proteasome and destroyed. Model systems have been established in mammalian cells to study the export of misfolded ER a of the transmembrane a is targeted for proteasomal C. S. R. Cell. Full Text PDF PubMed Scopus Google Scholar). of type I membrane proteins have been the human type I and US11 that to nascent MHC class I heavy in the ER and their export and by the proteasome T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. J. Nature. PubMed Scopus Google Scholar, L. J. PubMed Google Scholar). of the cell are exported and degraded J. H. Full Text Full Text PDF PubMed Scopus Google Scholar, H. G. S. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). degradation of proteins in the ER has been via the export of of the protein C. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, D. J. S. D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). are of ER proteins for ER quality control is in the folding and of proteins that to M. L. 2000; 1: PubMed Scopus Google Scholar, M. L. 2002; PubMed Scopus Google Scholar). of type I targeted for degradation in mammalian cells has been as a process and I heavy are in and US11-expressing a of and degradation can be through the of proteasome T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of class I heavy by cytosolic peptide occurs of the to the in cells that have been with proteasome inhibitor T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). MHC class I heavy as intermediates before degradation D. Mol. Biol. Cell. PubMed Scopus Google Scholar, C. H. J. Cell Biol. 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EMBO J. 2006; PubMed Scopus Google that the by US11 likely by the cell to export misfolded ER The study US11 as an ER dislocation and proteasome degradation substrate and the of proteasome inhibition the dislocation of glycosylated US11 molecules that are modified with ubiquitin of proteasome deglycosylated US11 intermediates with a proteasome suggesting that proteasome the final steps of US11 removal from the ER The define the dislocation and demonstrate that US11 and deglycosylation before extraction from the ER Ubiquitination is by deglycosylation and complete dislocation. and and US11 cells in modified with and in a cells to and in to class I heavy and from H. of that to and of The C. J. Google from dehydrogenase from and from Cell and US11 from the of in which the L. D. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). of the with for into and into the which as a US11 or with the into cells via a to as L. D. J. Biol. Chem. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar). cells a cell Cell and by and in of by for in with and cells in of and by for US11 with and cells for cells and with The polypeptides from the cells with and by Proteins by and US11 cells by and for the to and carried out as cell carried out by cells in of by of and and peptide as the in by the addition of for or for peptide and of or of peptide N-glycanase. carried out for of and and US11 cells in modified for carried out with of for in the of proteasome cells with for the in modified an of and for the by for and in of for with The cell by the addition of of of and with of for cells through for and for US11 polypeptides as The by and the in the The exposed to for or polypeptides of MHC class I molecules in and cells the The cells with and a The data The of class I molecules are by of cell of The of cells by the cell the to membrane to or peptide for to with and US11 polypeptides as The with isoelectric focusing and a for H. J. B. H. D. in Scholar). the for in to membrane, and to Subcellular cells or with for and in and and a with cells and for and the with for for from steps in and the an a final of and in and US11 in a The degradation of type I membrane is a process that membrane and cytosolic protein The of the proteasome to the extraction of degradation substrates is not well The study proteasome inhibition dislocation of the type I ER membrane protein the degradation of US11 to the of US11 cells and cells that US11 with for by a of to US11 from cell the and The and by US11 is a with a of US11 molecules not from US11 polypeptides from US11 cells the of the and from the of the US11 that has a of The of US11 molecules the of the that US11 targeted for The of US11 molecules is of that for ER degradation T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, J. H. Full Text Full Text PDF PubMed Scopus Google suggesting that US11 may be targeted for proteasomal degradation. this and US11 cells with or the proteasome inhibitor with for and for to US11 from cell the and to and US11 not from and US11 cells with the of US11 the in with However, US11 cells with proteasome of US11 the a species from the US11 from the by the Treatment with proteasome in the accumulation of deglycosylated class I heavy in US11-expressing cells T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of a US11 proteasome inhibition that deglycosylated US11 molecules may data demonstrate that US11 targeted for proteasomal of proteins may be a by to a ER stress response by of the US11 in the host as US11 has been a ER stress response B. N. A. L. H. J. PubMed Scopus Google Scholar). US11 major between US11 degradation and US11-mediated class I degradation is the of the US11-expressing class I heavy are degraded with a of T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google whereas the of US11 is The of degradation intermediates is likely the of proteasome inhibition and the of dislocation. that for accumulation of US11 degradation US11 from US11 cells that been with for to Treatment with a of proteasome inhibitor for a of not to cells not and by US11 not from cells of glycosylated US11 from US11 cells with proteasome with of glycosylated US11 from cells for and a from US11 cells with for and and accumulation of glycosylated and deglycosylated US11 species of proteasome inhibition is of US11 degradation polypeptides of proteasomal inhibition by molecules in the by and are likely US11 molecules modified with US11 species are modified with ubiquitin, as by of US11 and US11 from cells by a of to that in the be the that the to the US11 species data that of proteasome inhibition are to US11 degradation is an ER resident that a N-linked glycan T. J. PubMed Google Scholar, A. D. EMBO J. PubMed Scopus Google Scholar). of the N-linked glycan by the cytosolic enzyme peptide dislocation result in a the US11 species proteasome inhibition a deglycosylated degradation this US11 molecules with a enzyme that asparagine-linked from N. T. J. Biol. Chem. Full Text PDF Google Scholar). and US11 cells or with in of the to Cell via and US11, class I heavy and proteins by an their that of protein the US11 not in cell from US11-expressing a glycosylated of US11 to in polypeptides the glycosylated of US11 and the species in US11 cells with with glycosylated US11 has a to the species suggesting that the species likely a deglycosylated US11 intermediate. data demonstrate that the accumulation of a US11 species can be used as a for the dislocation the of proteasome MHC class I heavy by chain I heavy through the ER and where their N-linked glycans modified by G. H. R. H. H. J. Cell Biol. PubMed Scopus Google Scholar, J. G. H. J. Cell Biol. PubMed Scopus Google Scholar). class I heavy by and cells with a of class I heavy in the ER to the inhibition of proteasomal peptide This is by the of a of class I heavy to However, the of class I heavy in cells has the to and class I heavy degraded in a and not in US11-expressing cells and proteasome by with class I heavy as deglycosylated class I heavy proteasome inhibition of US11-expressing isoelectric focusing in with to that the US11 species a glycan in deglycosylated of US11 of by peptide in the of to T. L. M. H. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar, D. M. J. Nature. PubMed Scopus Google whereas not the of the from US11 cells or with in and or with and or peptide and US11 and the a isoelectric focusing a US11 cells with in and US11 polypeptides to with or peptide and by The US11 polypeptides to and The glycosylated US11 protein as a with a of and of and Treatment of US11 cell with not the or of glycosylated US11 US11 to as a deglycosylated intermediate the US11 from peptide an as to US11 polypeptides as deglycosylated species by US11 molecules from cells as that to glycosylated and deglycosylated of the Treatment with not the of US11 polypeptides by whereas US11 as a species by US11 from the with peptide the as the deglycosylated US11 intermediate demonstrate that US11 deglycosylated by cytosolic enzyme peptide as exported from the ER to the cytosol the dislocation US11 to I for US11 is targeted for proteasomal degradation in a to class I heavy is that that US11 and heavy are in a this a US11 that is of class I molecules for D. H. Mol. Biol. 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Cell. 14: PubMed Scopus Google data that class I heavy chain degradation is not a for US11 and are targeted for proteasomal and cells with the I by with to by and the of MHC class I molecules with to cell cells with are by a and cells with for or by in US11 molecules by with by and to an and deglycosylated US11 are and cells with for and in US11 proteins with and by by in the of US11 to of US11 to extraction from the ER US11 a in region that be a for the cytosolic of is critical for the initiation of US11 a where to ability to the of class I heavy by an folded class I molecules cells cell class I molecules to to US11 with class I in cells of class I degradation in cells by where deglycosylated class I heavy in cells not demonstrate that is and can class I molecules to We itself targeted for proteasomal degradation. 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Full Text Full Text PDF PubMed Scopus Google Scholar). by the of the glycosylated US11 US11 proteins modified with multiple data demonstrate that a of glycosylated US11 modified with ubiquitin integrated in the ER is not to dislocation and the of glycosylated that have exposed to the cytosol dislocation. this and molecules from cells with and to of and and and in the of and species to and and that from the and and and is that the of US11 that a US11 that has been ubiquitinated The accumulation of glycosylated and ubiquitinated and that in the that molecules modified the This that the region of US11 to the cytosol and modified with ubiquitin before complete deglycosylation and extraction from the ER data demonstrate that US11 molecules in the ER proteasomal a of the of ER substrates is a process that the dislocation of substrates the ER membrane by their proteasomal degradation in the that proteasome inhibition causes an accumulation of ubiquitinated US11 polypeptides and deglycosylated degradation intermediates that the dislocation and degradation of US11 are and proteasome inhibition for the accumulation of dislocation the deglycosylated US11 the membrane of glycosylated and deglycosylated US11 proteins proteasome inhibition of with causes the of proteins from the membrane T. J. PubMed Google Scholar). a within the is to this can be used to the US11 is integrated within the membrane or has been from the membrane that from US11 cells or with proteasome inhibitor a by a to and membrane D. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The or with to proteins from the The by a to the membrane from the cytosolic The and by and to US11, and is a ER protein that a in protein D. S. Nature. PubMed Scopus Google and can be used as a membrane protein is a cytosolic protein that with the ER membrane S. M. Trends Cell Biol. PubMed Google and can the of The membrane proteins US11 and in the membrane of and and and Examination of the that not of the protein with the membrane as by in the and and However, with to be from the into the cytosolic and and that for the of proteins from the US11 cells with proteasome in the membrane of and US11 in the and a result with the of a membrane the a of the deglycosylated US11 protein as a membrane protein and whereas a of deglycosylated US11 in the cytosolic with a in the of deglycosylated US11 as a cytosolic The of by the of in the that in the of proteasome deglycosylated US11 polypeptides in the ER membrane and are unable to be into the demonstrate that proteasome inhibition the accumulation of unique degradation intermediates and supports a model in which proteasome is involved in the final steps of US11 from the ER membrane, of peptide and before into the cytosol and degradation. degradation of US11 can be as a to define dislocation and degradation of ER type I of US11 degradation through of proteasome resulted in the accumulation of ubiquitinated and deglycosylated US11 The of degradation intermediates the of substrate extraction into the cytosol and for the of an model of ER dislocation The US11 is a type I with a transmembrane and The protein is modified with an N-linked glycan and and A. D. EMBO J. PubMed Scopus Google Scholar). The initiation of US11 degradation with the dislocation of US11 the ER the ER proteins or that the extraction of US11 have not been chaperones and transmembrane proteins such as in the dislocation of US11 the membrane Once glycosylated US11 polypeptides are ubiquitinated and The is a likely for However, the US11 to be targeted for degradation and can be modified with ubiquitin by a cellular E3 L. PubMed Scopus Google and L. J. Cell Biol. 2007; PubMed Scopus Google can be for E3 and US11 a in the The that this ubiquitin have been for E3 ligases. We that US11 is recognized as a degradation substrate and of by cellular machinery in a to a misfolded a model in which the US11 is before and ubiquitinated by a cellular E3 the ubiquitinated US11 species an N-linked the of US11 molecules that the region the N-linked glycan is within the ER are US11 US11 be through the membrane in a This is likely not the deglycosylated US11 integrated within the ER membrane with This that the transmembrane region of US11 is the to be out of the The US11 between and the transmembrane region are the membrane into the This is by the of multiple that can be The of US11 is the membrane, and the is modified with ubiquitin R. I. T. U. A. A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). is not to with the of the US11 the the that the is the ER membrane in a The model of dislocation and of US11 is by the accumulation of ubiquitinated in the of in B. T. Mol. Cell. 2004; 14: Full Text Full Text PDF PubMed Scopus Google Scholar). 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J. 2000; 14: PubMed Scopus Google that the proteasome in the dislocation of a of cellular The data into the of type I dislocation through the of degradation The of the of the proteasome in this process to be We for of the and by H.