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Live Cell Imaging of Protein Dislocation from the Endoplasmic Reticulum*

  • Yongwang Zhong
    Affiliations
    Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
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  • Shengyun Fang
    Correspondence
    To whom correspondence should be addressed: Center for Biomedical Engineering and Technology, Department of Physiology, University of Maryland School of Medicine, BioMET, 725 W Lombard St. Baltimore, MD. Tel.: 410-706-2220
    Affiliations
    Center for Biomedical Engineering and Technology and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201
    Search for articles by this author
  • Author Footnotes
    * This work was supported by NSF Grant 1120833 (to S. F.).
    This article contains supplemental Movies S1–S5 and Fig. S1.
Open AccessPublished:June 21, 2012DOI:https://doi.org/10.1074/jbc.M112.381798
      Misfolded proteins in the endoplasmic reticulum (ER) are dislocated to the cytosol to be degraded by the proteasomes. Various plant and bacterial toxins and certain viruses hijack this dislocation pathway to exert their toxicity or to infect cells. In this study, we report a dislocation-dependent reconstituted GFP (drGFP) assay that allows, for the first time, imaging proteins dislocated from the ER lumen to the cytosol in living cells. Our results indicate that both luminal and membrane-spanning ER proteins can be fully dislocated from the ER to the cytosol. By combining the drGFP assay with RNAi or chemical inhibitors of proteins in the Hrd1 ubiquitin ligase complex, we demonstrate that the Sel1L, Hrd1, p97/VCP, and importin β proteins are required for the dislocation of misfolded luminal α-1 antitrypsin. The strategy described in this work is broadly applicable to the study of other types of transmembrane transport of proteins and likely also of viruses and toxins in living cells.

      Introduction

      Proteins destined for the secretory pathway are inserted into the membrane or lumen of the endoplasmic reticulum (ER),
      The abbreviations used are: ER
      endoplasmic reticulum
      drGFP
      dislocation-dependent reconstituted GFP
      ERAD
      ER-associated degradation
      NHK
      null Hong Kong variant of α-1-antitrypsin
      ATM
      wild type α-1-antitrypsin
      PNGase F
      peptide: N-glycosidase F
      DBeQ
      N2,N4-dibenzylquinazoline-2,4-diamine
      IPZ
      importazole
      RFU
      relative fluorescence unit.
      where they are processed and folded into their native conformation. Misfolded or unassembled proteins are retained in the ER and transported to the cytosol for proteasomal degradation, a process known as ER-associated protein degradation (ERAD) (
      • Buchberger A.
      • Bukau B.
      • Sommer T.
      Protein quality control in the cytosol and the endoplasmic reticulum: brothers in arms.
      ,
      • Vembar S.S.
      • Brodsky J.L.
      One step at a time: endoplasmic reticulum-associated degradation.
      ). ERAD protects cells against the detrimental effects of ER stress and regulates many essential cellular functions, such as sterol homeostasis and calcium signaling (
      • Hampton R.Y.
      • Garza R.M.
      Protein quality control as a strategy for cellular regulation: lessons from ubiquitin-mediated regulation of the sterol pathway.
      ,
      • Goldstein J.L.
      • DeBose-Boyd R.A.
      • Brown M.S.
      Protein sensors for membrane sterols.
      ,
      • Lu J.P.
      • Wang Y.
      • Sliter D.A.
      • Pearce M.M.
      • Wojcikiewicz R.J.
      RNF170 protein, an endoplasmic reticulum membrane ubiquitin ligase, mediates inositol 1,4,5-trisphosphate receptor ubiquitination and degradation.
      ).
      The ER-to-cytosol transport, called dislocation or retrotranslocation, is one of the key steps in ERAD because the ubiquitin-proteasome system is localized in the cytosol (
      • Wiertz E.J.
      • Jones T.R.
      • Sun L.
      • Bogyo M.
      • Geuze H.J.
      • Ploegh H.L.
      The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.
      ,
      • Sommer T.
      • Jentsch S.
      A protein translocation defect linked to ubiquitin conjugation at the endoplasmic reticulum.
      ,
      • Ward C.L.
      • Omura S.
      • Kopito R.R.
      Degradation of CFTR by the ubiquitin-proteasome pathway.
      ,
      • Jensen T.J.
      • Loo M.A.
      • Pind S.
      • Williams D.B.
      • Goldberg A.L.
      • Riordan J.R.
      Multiple proteolytic systems, including the proteasome, contribute to CFTR processing.
      ,
      • McCracken A.A.
      • Brodsky J.L.
      Assembly of ER-associated protein degradation in vitro: dependence on cytosol, calnexin, and ATP.
      ,
      • Hiller M.M.
      • Finger A.
      • Schweiger M.
      • Wolf D.H.
      ER degradation of a misfolded luminal protein by the cytosolic ubiquitin-proteasome pathway.
      ). Various plant and bacterial toxins and certain viruses hijack the dislocation process to reach the cytosol, where they may exert their cytotoxicity or effectively infect cells (
      • Tsai B.
      • Rapoport T.A.
      Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1.
      ,
      • Spooner R.A.
      • Watson P.D.
      • Marsden C.J.
      • Smith D.C.
      • Moore K.A.
      • Cook J.P.
      • Lord J.M.
      • Roberts L.M.
      Protein disulfide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum.
      ,
      • Yu M.
      • Haslam D.B.
      Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3.
      ,
      • Kothe M.
      • Ye Y.
      • Wagner J.S.
      • De Luca H.E.
      • Kern E.
      • Rapoport T.A.
      • Lencer W.I.
      Role of p97 AAA-ATPase in the retrotranslocation of the cholera toxin A1 chain, a non-ubiquitinated substrate.
      ,
      • Schelhaas M.
      • Malmström J.
      • Pelkmans L.
      • Haugstetter J.
      • Ellgaard L.
      • Grünewald K.
      • Helenius A.
      Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells.
      ). In addition, growing evidence suggests that certain ER-localized proteins, such as the EGF receptor, ErbB2, calreticulin, Nrf1, and OS9, dislocate first to the cytosol and then are imported into the nucleus when they function as transcriptional regulators (
      • Lin S.Y.
      • Makino K.
      • Xia W.
      • Matin A.
      • Wen Y.
      • Kwong K.Y.
      • Bourguignon L.
      • Hung M.C.
      Nuclear localization of EGF receptor and its potential new role as a transcription factor.
      ,
      • Liao H.J.
      • Carpenter G.
      Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression.
      ,
      • Wang Y.N.
      • Yamaguchi H.
      • Huo L.
      • Du Y.
      • Lee H.J.
      • Lee H.H.
      • Wang H.
      • Hsu J.M.
      • Hung M.C.
      The translocon Sec61β localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus.
      ,
      • Afshar N.
      • Black B.E.
      • Paschal B.M.
      Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol.
      ,
      • Baek J.H.
      • Mahon P.C.
      • Oh J.
      • Kelly B.
      • Krishnamachary B.
      • Pearson M.
      • Chan D.A.
      • Giaccia A.J.
      • Semenza G.L.
      OS-9 interacts with hypoxia-inducible factor 1alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1α.
      ,
      • Steffen J.
      • Seeger M.
      • Koch A.
      • Krüger E.
      Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop.
      ). However, the mechanisms underlying dislocation remain poorly understood.
      The development of an effective assay for dislocation should facilitate research on this important process. Dislocation is typically analyzed by biochemical approaches. One method is to detect the cytosolic localization of dislocated substrate proteins by subcellular fractionation and immunoblotting (
      • Wiertz E.J.
      • Jones T.R.
      • Sun L.
      • Bogyo M.
      • Geuze H.J.
      • Ploegh H.L.
      The human cytomegalovirus US11 gene product dislocates MHC class I heavy chains from the endoplasmic reticulum to the cytosol.
      ,
      • Tiwari S.
      • Weissman A.M.
      Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitin-conjugating enzymes (E2s).
      ). Another approach is to analyze dislocation indirectly by detecting substrate deglycosylation (for glycoproteins) or substrate ubiquitination (for luminal substrates), events that occur only when the substrates reach the cytosol (
      • Tiwari S.
      • Weissman A.M.
      Endoplasmic reticulum (ER)-associated degradation of T cell receptor subunits. Involvement of ER-associated ubiquitin-conjugating enzymes (E2s).
      ,
      • Nakatsukasa K.
      • Huyer G.
      • Michaelis S.
      • Brodsky J.L.
      Dissecting the ER-associated degradation of a misfolded polytopic membrane protein.
      ,
      • Ernst R.
      • Mueller B.
      • Ploegh H.L.
      • Schlieker C.
      The otubain YOD1 is a deubiquitinating enzyme that associates with p97 to facilitate protein dislocation from the ER.
      ,
      • Wang Q.
      • Liu Y.
      • Soetandyo N.
      • Baek K.
      • Hegde R.
      • Ye Y.
      A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation.
      ,
      • Greenblatt E.J.
      • Olzmann J.A.
      • Kopito R.R.
      Derlin-1 is a rhomboid pseudoprotease required for the dislocation of mutant α-1 antitrypsin from the endoplasmic reticulum.
      ). However, these methods are not always effective because the dislocation of certain substrates is tightly coupled with their ubiquitination and subsequent proteasomal degradation (
      • Nakatsukasa K.
      • Brodsky J.L.
      The recognition and retrotranslocation of misfolded proteins from the endoplasmic reticulum.
      ). The complexity of dislocation processes also requires more efficient assays. Nearly ninety proteins in mammalian cells are involved in ERAD or have high-confidence interactions with ERAD machineries (
      • Wang Q.
      • Liu Y.
      • Soetandyo N.
      • Baek K.
      • Hegde R.
      • Ye Y.
      A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation.
      ,
      • Mueller B.
      • Klemm E.J.
      • Spooner E.
      • Claessen J.H.
      • Ploegh H.L.
      SEL1L nucleates a protein complex required for dislocation of misfolded glycoproteins.
      ,
      • Jo Y.
      • Sguigna P.V.
      • DeBose-Boyd R.A.
      Membrane-associated ubiquitin ligase complex containing gp78 mediates sterol-accelerated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.
      ,
      • Christianson J.C.
      • Olzmann J.A.
      • Shaler T.A.
      • Sowa M.E.
      • Bennett E.J.
      • Richter C.M.
      • Tyler R.E.
      • Greenblatt E.J.
      • Wade Harper J.
      • Kopito R.R.
      Defining human ERAD networks through an integrative mapping strategy.
      ,
      • Zhong Y.
      • Wang Y.
      • Yang H.
      • Ballar P.
      • Lee J.G.
      • Ye Y.
      • Monteiro M.J.
      • Fang S.
      Importin β interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant α1-antitrypsin.
      ). The actual number of ERAD components could easily be over a hundred, as the actual components do not necessarily have to interact with the ERAD machinery with high affinity. These ERAD components are organized into various complexes that are centered on one of the many proven or potential ERAD E3 ubiquitin ligases, which dispose of misfolded proteins with various characteristics (
      • Carvalho P.
      • Goder V.
      • Rapoport T.A.
      Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.
      ,
      • Denic V.
      • Quan E.M.
      • Weissman J.S.
      A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.
      ,
      • Bernasconi R.
      • Galli C.
      • Calanca V.
      • Nakajima T.
      • Molinari M.
      Stringent requirement for HRD1, SEL1L, and OS-9/XTP3-B for disposal of ERAD-LS substrates.
      ). Moreover, it is possible that a given E3 can organize different complexes that perform different roles in different cells (
      • Bagola K.
      • Mehnert M.
      • Jarosch E.
      • Sommer T.
      Protein dislocation from the ER.
      ,
      • Eura Y.
      • Yanamoto H.
      • Arai Y.
      • Okuda T.
      • Miyata T.
      • Kokame K.
      Derlin-1 deficiency is embryonic lethal, Derlin-3 deficiency appears normal, and Herp deficiency is intolerant to glucose load and ischemia in mice.
      ). It would be difficult and tedious to study the large number of potential regulators in many different dislocation complexes by the currently available biochemical methods. Therefore, the establishment of an efficient live cell assay for dislocation could significantly expedite the study of the molecular mechanisms of dislocation.
      In this study, we report a novel split-GFP-based dislocation assay. In this assay, dislocation leads to the reconstitution of GFP fluorescence from its fragments. The reconstituted GFP serves as a reporter for the localization and quantity of dislocated substrates in living cells. By combining this approach with RNAi and chemical inhibitors of proteins in ERAD complexes, we demonstrate the feasibility of this assay for analyzing the mechanisms that underlie the dislocation of various types of substrates. Moreover, the strategy described herein should be widely applicable to the study of other types of transmembrane transport of proteins in living cells.

      DISCUSSION

      Based on the reassembly property of the split-GFP system (
      • Cabantous S.
      • Terwilliger T.C.
      • Waldo G.S.
      Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein.
      ), we have established a dislocation-dependent reconstituted GFP (drGFP) assay. The usefulness of the drGFP assay for studying dislocation was validated by various biochemical approaches. More importantly, we have used this assay to make several important observations. First, we demonstrated that both the luminal substrate NHK and the membrane-spanning substrate CD3δ could be fully dislocated to the cytosol. Second, we found that importin β cooperates with p97/VCP to regulate NHK dislocation. Third, we showed that Hrd1 is not only functions as an E3 ubiquitin ligase but also plays an essential role in NHK dislocation. It may act as a dislocation channel, similar to its yeast counterpart (
      • Carvalho P.
      • Stanley A.M.
      • Rapoport T.A.
      Retrotranslocation of a misfolded luminal ER protein by the ubiquitin-ligase Hrd1p.
      ). Fourth, we found that the Sel1L protein, the luminal substrate receptor, is also essential for NHK dislocation, suggesting that Sel1L may play a key role in the initiation of dislocation. Fifth, we showed that proteasome activity is not required for dislocation in living cells, which is consistent with previous reports using in vitro reconstitution assays (
      • Lee R.J.
      • Liu C.W.
      • Harty C.
      • McCracken A.A.
      • Latterich M.
      • Römisch K.
      • DeMartino G.N.
      • Thomas P.J.
      • Brodsky J.L.
      Uncoupling retro-translocation and degradation in the ER-associated degradation of a soluble protein.
      ). This finding is supported by the fact that the drGFP signal continuously increases when proteasome activity is inhibited. We can envision that this new assay will be appropriate for studying several novel aspects of dislocation that are otherwise difficult or not possible to study with the current biochemical methods. For example, drGFP can be used to rapidly monitor and quantify dislocation in living cells. It specifically enables the study of the fate, localization, and solubility (aggregation) of dislocated substrates under physiological and pathological conditions. More importantly, it can be combined with large-scale RNAi and compound screens for proteins and chemical modulators of dislocation of a given specific substrate protein. This assay has the potential to be adopted for studying the dislocation of all luminal substrates and all membrane substrates that have at least one terminus (N or C terminus) in the ER lumen. The luminal terminus of membrane-spanning substrates can serve as the site for S11 tagging. However, as with other biological assays, the drGFP assay has limitations. For example, a ubiquitination site too close to the S11 tag may interfere with the reassembly of S11 and S1-10. Thus, one should be cautious when an unexplainable negative result is obtained with the drGFP assay. Substrate folding state could be another factor that affects the drGFP assay. Misfolded substrates may embed S11 and block GFP reassembly. However, this may not be a concern; increasing evidence indicates that substrates dislocate in unfolded or partially unfolded states (
      • Wang Q.
      • Liu Y.
      • Soetandyo N.
      • Baek K.
      • Hegde R.
      • Ye Y.
      A ubiquitin ligase-associated chaperone holdase maintains polypeptides in soluble states for proteasome degradation.
      ,
      • Bagola K.
      • Mehnert M.
      • Jarosch E.
      • Sommer T.
      Protein dislocation from the ER.
      ,
      • Nishikawa S.I.
      • Fewell S.W.
      • Kato Y.
      • Brodsky J.L.
      • Endo T.
      Molecular chaperones in the yeast endoplasmic reticulum maintain the solubility of proteins for retrotranslocation and degradation.
      ,
      • Claessen J.H.
      • Ploegh H.L.
      BAT3 guides misfolded glycoproteins out of the endoplasmic reticulum.
      ). In addition, S11 and S1-10 reassembly is time- and concentration-dependent, which could decrease the sensitivity of the drGFP assay for dislocation. Nonetheless, the present study indicates that the drGFP assay is a simple and reliable assay and should significantly expedite the study of dislocation in ERAD.
      The strategy described in this article should be widely applicable to the study of other transmembrane transport of proteins in living cells, such as protein transport across the membranes of mitochondria, Golgi, peroxisomes and lysosome, as well as plasma and nuclear membranes. Moreover, it is well known that certain toxins, such as ricin and cholera toxin, and viruses, such as Simian Virus 40 (SV40), enter cells through endocytosis followed by vesicular transport to the lumen of the ER. These toxins and viruses then co-opt the dislocation process to reach the cytosol, where they exert their cytotoxicity or infect cells (
      • Tsai B.
      • Rapoport T.A.
      Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1.
      ,
      • Spooner R.A.
      • Watson P.D.
      • Marsden C.J.
      • Smith D.C.
      • Moore K.A.
      • Cook J.P.
      • Lord J.M.
      • Roberts L.M.
      Protein disulfide-isomerase reduces ricin to its A and B chains in the endoplasmic reticulum.
      ,
      • Yu M.
      • Haslam D.B.
      Shiga toxin is transported from the endoplasmic reticulum following interaction with the luminal chaperone HEDJ/ERdj3.
      ,
      • Kothe M.
      • Ye Y.
      • Wagner J.S.
      • De Luca H.E.
      • Kern E.
      • Rapoport T.A.
      • Lencer W.I.
      Role of p97 AAA-ATPase in the retrotranslocation of the cholera toxin A1 chain, a non-ubiquitinated substrate.
      ,
      • Schelhaas M.
      • Malmström J.
      • Pelkmans L.
      • Haugstetter J.
      • Ellgaard L.
      • Grünewald K.
      • Helenius A.
      Simian Virus 40 depends on ER protein folding and quality control factors for entry into host cells.
      ). We envision that dislocation of these toxins or viruses can be readily monitored in living cells using S11 fused with the toxins or with a viral surface protein. In addition, many proteins, such as the EGF receptor, ErbB2, Calreticulin, OS9, and Nrf1, can translocate from the ER to the nucleus when they function as transcriptional regulators and regulate important cellular events, such as tumorigenesis and oxidative defense (
      • Lin S.Y.
      • Makino K.
      • Xia W.
      • Matin A.
      • Wen Y.
      • Kwong K.Y.
      • Bourguignon L.
      • Hung M.C.
      Nuclear localization of EGF receptor and its potential new role as a transcription factor.
      ,
      • Liao H.J.
      • Carpenter G.
      Role of the Sec61 translocon in EGF receptor trafficking to the nucleus and gene expression.
      ,
      • Wang Y.N.
      • Yamaguchi H.
      • Huo L.
      • Du Y.
      • Lee H.J.
      • Lee H.H.
      • Wang H.
      • Hsu J.M.
      • Hung M.C.
      The translocon Sec61β localized in the inner nuclear membrane transports membrane-embedded EGF receptor to the nucleus.
      ,
      • Afshar N.
      • Black B.E.
      • Paschal B.M.
      Retrotranslocation of the chaperone calreticulin from the endoplasmic reticulum lumen to the cytosol.
      ,
      • Baek J.H.
      • Mahon P.C.
      • Oh J.
      • Kelly B.
      • Krishnamachary B.
      • Pearson M.
      • Chan D.A.
      • Giaccia A.J.
      • Semenza G.L.
      OS-9 interacts with hypoxia-inducible factor 1alpha and prolyl hydroxylases to promote oxygen-dependent degradation of HIF-1α.
      ,
      • Steffen J.
      • Seeger M.
      • Koch A.
      • Krüger E.
      Proteasomal degradation is transcriptionally controlled by TCF11 via an ERAD-dependent feedback loop.
      ). It is generally thought that these proteins first utilize the ERAD dislocation machinery to enter the cytosol and are then imported into the nucleus. However, the underlying mechanisms of this pathway have not yet been established. Application of the drGFP assay should significantly facilitate research in this area. More importantly, the drGFP assays for toxins, viruses and oncogenes, once established, could serve as a simple platform for high-throughput screening of small molecule inhibitors, facilitating dislocation-targeted drug discovery for these specific targets.

      Acknowledgments

      We thank Dr. Rebecca Heald for the generous gift of importazole, Dr. Mariusz Karbowski for access to microscope for live cell imaging, and Dr. Mervyn J. Monteiro for helpful discussions.

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