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Importin β Interacts with the Endoplasmic Reticulum-associated Degradation Machinery and Promotes Ubiquitination and Degradation of Mutant α1-Antitrypsin*

Open AccessPublished:August 08, 2011DOI:https://doi.org/10.1074/jbc.M111.272906
      The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.

      Introduction

      Many newly synthesized proteins in the endoplasmic reticulum (ER)
      The abbreviations used are: ER
      endoplasmic reticulum
      ERAD
      ER-associated degradation
      A1AT
      α-1-antitrypsin
      NHK
      null Hong Kong variant of α-1-antitrypsin
      IP
      immunoprecipitation
      IB
      immunoblotting
      VIMP
      p97/valosin-containing protein-interacting membrane protein
      NTF2
      nuclear transport factor 2
      Ni-NTA
      nickel-nitrilotriacetic acid.
      fail to fold properly because of transcriptional and translational errors or imbalanced production of accessory subunits (
      • Vembar S.S.
      • Brodsky J.L.
      ,
      • Hirsch C.
      • Gauss R.
      • Horn S.C.
      • Neuber O.
      • Sommer T.
      ). Additionally, pathogenic conditions, such as genetic mutations, hypoxia, oxidative stress, ischemia, and disturbance of calcium homeostasis, can also cause proteins to misfold in the ER (
      • Kim I.
      • Xu W.
      • Reed J.C.
      ,
      • Zhang K.
      • Kaufman R.J.
      ,
      • Sato B.K.
      • Schulz D.
      • Do P.H.
      • Hampton R.Y.
      ). Fortunately, cells have evolved protein quality control systems that can efficiently eliminate misfolded proteins from the ER before they wreak havoc on cells. A major mechanism that recognizes and degrades misfolded and unassembled ER proteins at the ER is the ER-associated degradation (ERAD) pathway (
      • Vembar S.S.
      • Brodsky J.L.
      ,
      • Hirsch C.
      • Gauss R.
      • Horn S.C.
      • Neuber O.
      • Sommer T.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Hampton R.Y.
      ,
      • Hebert D.N.
      • Bernasconi R.
      • Molinari M.
      ), which exports misfolded ER proteins into the cytosol for degradation by the ubiquitin proteasome system.
      ER luminal chaperones and lectins recognize and deliver ERAD substrates to membrane-anchored protein complexes that form putative protein-conducting channels from which the substrates are subsequently retrotranslocated into the cytosol (
      • Hebert D.N.
      • Bernasconi R.
      • Molinari M.
      ,
      • Wang S.
      • Ng D.T.
      ). Each of these retrotranslocation complexes usually contains one or more membrane-bound ubiquitin ligases (E3s), which ubiquitinate ERAD substrates en route to the cytosol. In budding yeast, the Hrd1p E3 complex degrades substrates whose lesions reside in either the transmembrane domain or lumen of the ER, whereas the other complex containing the Doa10p E3 disposes of substrates with lesions in their cytosolic domains (
      • Carvalho P.
      • Goder V.
      • Rapoport T.A.
      ,
      • Denic V.
      • Quan E.M.
      • Weissman J.S.
      ). These ERAD complexes are conserved in mammalian cells, but as expected, the repertoire of E3s for mammalian ERAD is more complex. In addition to the Hrd1p and Doa10p orthologs (Hrd1 and gp78 for Hrd1p and TEB4 for Doa10p) (
      • Fang S.
      • Ferrone M.
      • Yang C.
      • Jensen J.P.
      • Tiwari S.
      • Weissman A.M.
      ,
      • Nadav E.
      • Shmueli A.
      • Barr H.
      • Gonen H.
      • Ciechanover A.
      • Reiss Y.
      ,
      • Amano T.
      • Yamasaki S.
      • Yagishita N.
      • Tsuchimochi K.
      • Shin H.
      • Kawahara K.
      • Aratani S.
      • Fujita H.
      • Zhang L.
      • Ikeda R.
      • Fujii R.
      • Miura N.
      • Komiya S.
      • Nishioka K.
      • Maruyama I.
      • Fukamizu A.
      • Nakajima T.
      ,
      • Kikkert M.
      • Doolman R.
      • Dai M.
      • Avner R.
      • Hassink G.
      • van Voorden S.
      • Thanedar S.
      • Roitelman J.
      • Chau V.
      • Wiertz E.
      ,
      • Hassink G.
      • Kikkert M.
      • van Voorden S.
      • Lee S.J.
      • Spaapen R.
      • van Laar T.
      • Coleman C.S.
      • Bartee E.
      • Früh K.
      • Chau V.
      • Wiertz E.
      ), several other membrane-bound E3s, such as RMA1/RNF5 (ring finger protein 5), RFP2 (Ret finger protein 2), and TRC8 (translocation in renal cancer from chromosome 8) have been implicated in ERAD (
      • Kostova Z.
      • Tsai Y.C.
      • Weissman A.M.
      ,
      • Mehnert M.
      • Sommer T.
      • Jarosch E.
      ). In addition, cytosolic E3s, including CHIP (C terminus of Hsc70-interacting protein) (
      • Meacham G.C.
      • Patterson C.
      • Zhang W.
      • Younger J.M.
      • Cyr D.M.
      ,
      • Connell P.
      • Ballinger C.A.
      • Jiang J.
      • Wu Y.
      • Thompson L.J.
      • Höhfeld J.
      • Patterson C.
      ), Parkin (
      • Imai Y.
      • Soda M.
      • Inoue H.
      • Hattori N.
      • Mizuno Y.
      • Takahashi R.
      ), and the SCF (Skpl-Cullin-F-box protein family) multisubunit E3 (
      • Yoshida Y.
      • Chiba T.
      • Tokunaga F.
      • Kawasaki H.
      • Iwai K.
      • Suzuki T.
      • Ito Y.
      • Matsuoka K.
      • Yoshida M.
      • Tanaka K.
      • Tai T.
      ), can also be recruited to the cytosolic surface of the ER to act on ERAD substrates.
      ERAD substrates have to be fully retrotranslocated or dislocated from the ER for elimination (
      • Vembar S.S.
      • Brodsky J.L.
      ,
      • Hirsch C.
      • Gauss R.
      • Horn S.C.
      • Neuber O.
      • Sommer T.
      ,
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Hampton R.Y.
      ,
      • Hebert D.N.
      • Bernasconi R.
      • Molinari M.
      ), which is mediated by a conserved cytosolic AAA ATPase termed p97 in mammals or Cdc48p in yeast (
      • Tsai B.
      • Ye Y.
      • Rapoport T.A.
      ,
      • Bays N.W.
      • Hampton R.Y.
      ). The mechanism underlying protein retrotranslocation is poorly defined. It is generally believed that substrates are retrotranslocated from the ER through one or a few proteineous channels (
      • Vembar S.S.
      • Brodsky J.L.
      ), although the identity of such channel has not been revealed. One channel candidate protein is the multispanning membrane protein, Derlin-1, based on its involvement in retrotranslocation of nascent major histocompatibility complex class I heavy chains (
      • Lilley B.N.
      • Ploegh H.L.
      ,
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ), pαF (
      • Wahlman J.
      • DeMartino G.N.
      • Skach W.R.
      • Bulleid N.J.
      • Brodsky J.L.
      • Johnson A.E.
      ), simian virus 40 (
      • Schelhaas M.
      • Malmström J.
      • Pelkmans L.
      • Haugstetter J.
      • Ellgaard L.
      • Grünewald K.
      • Helenius A.
      ), and cholera toxin (
      • Bernardi K.M.
      • Forster M.L.
      • Lencer W.I.
      • Tsai B.
      ). Alternative candidates include the multispanning E3 ligases such as gp78, Hrd1, and TEB4 (
      • Meusser B.
      • Hirsch C.
      • Jarosch E.
      • Sommer T.
      ,
      • Zhong X.
      • Shen Y.
      • Ballar P.
      • Apostolou A.
      • Agami R.
      • Fang S.
      ,
      • Carvalho P.
      • Stanley A.M.
      • Rapoport T.A.
      ). Upon emerging into the cytosol, substrates are ubiquitinated by membrane-associated E3s and subsequently dislocated into the cytosol by the p97/Cdc48 complex (
      • Ye Y.
      • Meyer H.H.
      • Rapoport T.A.
      ,
      • Nakatsukasa K.
      • Huyer G.
      • Michaelis S.
      • Brodsky J.L.
      ). The ER membrane-anchored Ubx domain proteins, Ubx2p in yeast and erasin/UbxD2 and/or UbxD8 in mammalian cells, have been shown to recruit p97/Cdc48 to the ER for retrotranslocation (
      • Neuber O.
      • Jarosch E.
      • Volkwein C.
      • Walter J.
      • Sommer T.
      ,
      • Lim P.J.
      • Danner R.
      • Liang J.
      • Doong H.
      • Harman C.
      • Srinivasan D.
      • Rothenberg C.
      • Wang H.
      • Ye Y.
      • Fang S.
      • Monteiro M.J.
      ,
      • Mueller B.
      • Klemm E.J.
      • Spooner E.
      • Claessen J.H.
      • Ploegh H.L.
      ). In addition, several additional ER membrane proteins, including Hrd1, gp78, VIMP (p97/valosin-containing protein-interacting membrane protein), Derlin-1, and Herp, also interact with p97 in mammalian cells (
      • Ye Y.
      • Shibata Y.
      • Yun C.
      • Ron D.
      • Rapoport T.A.
      ,
      • Ye Y.
      • Shibata Y.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Rapoport T.A.
      ,
      • Ballar P.
      • Shen Y.
      • Yang H.
      • Fang S.
      ,
      • Schulze A.
      • Standera S.
      • Buerger E.
      • Kikkert M.
      • van Voorden S.
      • Wiertz E.
      • Koning F.
      • Kloetzel P.M.
      • Seeger M.
      ), suggesting that recruitment of p97 to the ER might occur through a concerted effort of multiple proteins. ERAD substrates retrotranslocated by p97/Cdc48p are then subjected to deglycosylation and ubiquitin chain editing before being shuttled to the proteasome for degradation (
      • Park H.
      • Suzuki T.
      • Lennarz W.J.
      ,
      • Wang Q.
      • Li L.
      • Ye Y.
      ,
      • Ernst R.
      • Mueller B.
      • Ploegh H.L.
      • Schlieker C.
      ,
      • Ernst R.
      • Claessen J.H.
      • Mueller B.
      • Sanyal S.
      • Spooner E.
      • van der Veen A.G.
      • Kirak O.
      • Schlieker C.D.
      • Weihofen W.A.
      • Ploegh H.L.
      ).
      To identify additional factors involved in ERAD, we searched for proteins that bind to the p97 receptor VIMP. Through this approach, we identified importin β as a new protein involved in ERAD. Importin β (also known as karyopherin β1 or kap95 in budding yeast) is a multifunctional protein that is known to mediate nuclear import of numerous proteins, act as a cytoplasmic chaperone that prevents aggregation for some proteins, and negatively regulate mitotic spindle assembly, nuclear membrane fusion, and nuclear pore complex formation (
      • Harel A.
      • Forbes D.J.
      ). During nuclear import, importin β binds cargo directly or utilizes importin α as an adaptor to bind its cargo proteins bearing a classical nuclear localization signal. The importin β-cargo complex is transported through the nuclear pore complex into the nucleus where RanGTP binds to importin β to induce cargo release. The importin β-RanGTP complex is then recycled back to the cytoplasm where RanGTP is dissociated from importin β and converted to RanGDP (
      • Cook A.
      • Bono F.
      • Jinek M.
      • Conti E.
      ). RanGDP is imported into the nucleus by NTF2 (nuclear transport factor 2) and then converted to RanGTP by RCC1 (regulator of chromosome condensation 1) (
      • Ribbeck K.
      • Lipowsky G.
      • Kent H.M.
      • Stewart M.
      • Görlich D.
      ,
      • Görlich D.
      • Seewald M.J.
      • Ribbeck K.
      ). The process results in RanGTP predominantly in the nucleus and RanGDP mainly in the cytoplasm (
      • Cook A.
      • Bono F.
      • Jinek M.
      • Conti E.
      ). Here we present evidence for a novel function of importin β in promoting ERAD. We show that importin β is an important component of a retrotranslocation machinery involved in ERAD and that it cooperates with Ran GTPase to promote ubiquitination and degradation of the luminal ERAD substrate, the null Hong Kong variant of α-1-antitrypsin (NHK).

      DISCUSSION

      In this study, we demonstrate a surprising connection between ERAD and importin β, a well known regulator of nucleocytoplasmic transport. Our in vivo and in vitro studies support the conclusion that importin β is required for ubiquitination of the luminal soluble ERAD substrate NHK. We demonstrated that siRNA-mediated knockdown of importin β reduces NHK ubiquitination. This phenotype can be recapitulated in an in vitro assay in which importin β is biochemically depleted from the cytosol and readdition of recombinant importin β to importin β-depleted cytosol restores the ubiquitination of NHK. By contrast, ubiquitination of the membrane ERAD substrate CD3δ increased in importin β knockdown cells. Importin β knockdown stabilizes both NHK and CD3δ. Therefore, diminished NHK ubiquitination is not likely to be resulted from a defect in the ubiquitin conjugating/deconjugating systems associated with the ER membrane. Otherwise, both luminal and membrane substrates should be affected in a similar way. Importin β is likely to act in a step required for the degradation of both substrates but only required for the ubiquitination of NHK. As a luminal ERAD substrate, NHK must first be retrotranslocated to the cytosolic side of the ER for ubiquitination because all known E3s involved in ERAD have their catalytic domains located in the cytosolic side. Thus, the ubiquitination levels of an ER luminal substrate, such as NHK, can be a faithful indicator of retrotranslocation when proteasomal degradation is inhibited. On the other hand, the membrane-bound substrates that contain cytosolically exposed domains, such as CD3δ, may be ubiquitinated directly on their cytosolic residues without the need of retrotranslocation. We propose that importin β may have a function in NHK retrotranslocation. Importin β may act first to promote extrusion of NHK into the cytosol for ubiquitination, after which p97 binds to the ubiquitin conjugates on NHK and extracts it from the translocation site for subsequent proteasomal degradation. Our data showed that importin β does not interact with p97, suggesting that they may not need physical interaction to cooperate in ERAD. However, we cannot exclude the possibility that importin β and p97 have weak or transient interaction during ERAD. Our study reveals association of importin β with several ERAD components in a retrotranslocation complex, including VIMP, Derlin-1, gp78, and Hrd1. Among these proteins, at least VIMP binds importin β directly. These interactions may recruit importin β to the ER and enable the latter to directly participate in retrotranslocation. In support of this possibility, a mutant of importin β (N297) that inhibits importin β association with the ER-enriched microsomes also inhibits ERAD of NHK.
      Structural studies show that in the absence of protein binding, importin β adopts an extended S-shaped superhelical architecture formed by HEAT repeats that consist of pairs of anti-parallel α-helices. The spring-like superhelical structure enables importin β to adapt its geometry to fit cargos of different sizes and shapes. When importin β binds to its cargo or the regulators Ran and importin α, it undergoes a drastic conformational change, which converts it to a compact spring-like form. This compact conformation stores energy, which is used for nuclear transport (
      • Zachariae U.
      • Grubmüller H.
      ). By analogy, importin β might alter between the two different energy states at the site of retrotranslocation, which could provide the necessary energy for protein retrotranslocation. Our results suggest that importin β interacts with RanGDP to facilitate ERAD, whereas RanGTP and importin α inhibit ERAD. We speculate that RanGDP is easier to be dissociated from importin β because of its weak affinity to the latter, and hence importin β in complex with RanGDP is easier to switch from the compact conformation to the extended conformation and release energy. It is conceivable that the conformational change in importin β, if it occurs within a retrotranslocation complex, may generate a localized force to regulate the gating of a retrotranslocation channel. By contrast, the high affinity binding of RanGTP or importin α to importin β may keep importin β in the compact conformation, which results in a blockage of retrotranslocation.
      Cytosolic localization of RanGDP and importin α may be pivotal to regulate the ERAD capacity. Previous studies reported that a significant amount of Ran was distributed to the cytoplasm from the nucleus in response to oxidative stress induced by H2O2 (
      • Miyamoto Y.
      • Saiwaki T.
      • Yamashita J.
      • Yasuda Y.
      • Kotera I.
      • Shibata S.
      • Shigeta M.
      • Hiraoka Y.
      • Haraguchi T.
      • Yoneda Y.
      ,
      • Yasuda Y.
      • Miyamoto Y.
      • Saiwaki T.
      • Yoneda Y.
      ). In contrast, H2O2-induced stress accumulates importin α, a potential inhibitor of ERAD, in the nucleus (
      • Miyamoto Y.
      • Saiwaki T.
      • Yamashita J.
      • Yasuda Y.
      • Kotera I.
      • Shibata S.
      • Shigeta M.
      • Hiraoka Y.
      • Haraguchi T.
      • Yoneda Y.
      ). Oxidative stress is known to cause overproduction of misfolded proteins in the ER. Therefore, the relocation of Ran and importin α under oxidative stress induced by H2O2 may represent a novel strategy to cope with cellular stress. Clearly, the unexpected interaction of importin β with ERAD machinery reveals a new layer of complexity for the regulation of ER quality control system in mammalian cells.

      Acknowledgments

      We thank Drs. Dirk Görlich, Richard Sifers, Karsten Weis, Reinhard Depping, Yun Qiu, Maria G. Masucci, and Wei Gu for providing plasmid constructs. We are grateful to Drs. Gong Li and Joe Kao for providing expert help in confocal microscopy.

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