1、Contents lists available at ScienceDirectSeminars in Cell&Developmental Biologyjournal homepage: protein degradation at the endoplasmic reticulum andnuclear envelopeAdrian B.Mehrtasha,Mark Hochstrasserb,a,aDepartment of Molecular,Cellular,&Developmental Biology,Yale University,New Haven,06520,CT,USA
2、bDepartment of Molecular Biophysics&Biochemistry,Yale University,New Haven,CT,06520,USAA R T I C L E I N F OKeywords:Protein degradationER-associated degradationProteasomeUbiquitinRetrotranslocationEndoplasmic reticulumA B S T R A C TNumerous nascent proteins undergo folding and maturation within th
3、e luminal and membrane compartments ofthe endoplasmic reticulum(ER).Despite the presence of various factors in the ER that promote protein folding,many proteins fail to properly fold and assemble and are subsequently degraded.Regulatory proteins in the ERalso undergo degradation in a way that is res
4、ponsive to stimuli or the changing needs of the cell.As in mostcellular compartments,the ubiquitin-proteasome system(UPS)is responsible for the majority of the degradationat the ERin a process termed ER-associated degradation(ERAD).Autophagic processes utilizing ubiquitin-likeprotein-conjugating sys
5、tems also play roles in protein degradation at the ER.The ER is continuous with thenuclear envelope(NE),which consists of the outer nuclear membrane(ONM)and inner nuclear membrane(INM).While ERAD is known also to occur at the NE,only some of the ERAD ubiquitin-ligation pathwaysfunction at the INM.Pr
6、otein degradation machineries in the ER/NE target a wide variety of substrates inmultiple cellular compartments,including the cytoplasm,nucleoplasm,ER lumen,ER membrane,and the NE.Here,we review the protein degradation machineries of the ER and NE and the underlying mechanisms dictatingrecognition a
7、nd processing of substrates by these machineries.1.IntroductionThe endoplasmic reticulum(ER)is a massive intracellular organellecomposed of a continuous membrane system that includes the periph-eral ER and nuclear membranes.The nuclear envelope(NE)is a doublelipid bilayer consisting of an outer nucl
8、ear membrane(ONM)and innernuclear membrane(INM)that encapsulates the nucleus.While the ONMis generally viewed as an extension of the peripheral ER,the INMcontains distinct physical characteristics along with a specific subset ofmembrane-residing proteins,albeit with some overlap 1.The ER hasmany fun
9、ctions in cellular regulation 2;perhaps the most prominentand conserved role of the ER is as the major site for protein synthesisand subsequent maturation of membrane and secreted proteins.Nu-merous factors present at the ER guide the proper folding and mod-ification of nascent proteins 3;neverthele
10、ss,many proteins fail tomature properly and need to be extracted from the ER and degraded.Additionally,protein levels must be carefully coordinated in responseto environmental cues,such as nutrient availability and proteotoxicstress.Protein modification by covalent ubiquitin addition and sub-sequent
11、 degradation by the proteasome,as well as autophagic processesthat utilize ubiquitin-like protein(Ubl)-conjugating systems are key tocontrolling protein levels both in the ER and elsewhere in the cell 4.2.The ubiquitin-proteasome systemProtein degradation is a closely regulated process that serves t
12、oeliminate short-lived regulatory proteins as well as misfolded proteins5.Failure to discard misfolded proteins often leads to their accumu-lation as protein aggregates,a common theme in the development ofmany degenerative diseases 6.The ubiquitin-proteasome system(UPS)is responsible for most select
13、ive protein degradation in eu-karyotes.In this system,proteins are marked for proteasomal de-gradation by the covalent attachment of ubiquitin to a protein,a pro-cess termed ubiquitylation 7,8.Ubiquitylation begins with an ATP-dependent reaction that is catalyzed by an E1 ubiquitin-activating en-zym
14、e,leading to a high-energy thioester linkage between the ubiquitinC-terminus(Gly76)and an active-site cysteine residue of the E1.Ubi-quitin is then transferred from the E1 to an active-site cysteine of aubiquitin-conjugating(E2)enzyme.Finally,in the most common me-chanism,an E3 ubiquitin ligase medi
15、ates the interaction between theE2ubiquitin and target protein,stimulating ubiquitin transfer to thetarget protein,usually to a lysine side chain(s)9.The E3s are thehttps:/doi.org/10.1016/j.semcdb.2018.09.013Received 7 August 2018;Received in revised form 26 September 2018;Accepted 27 September 2018
16、Corresponding author at:Department of Molecular Biophysics&Biochemistry,Yale University,New Haven,06520,CT,USA.E-mail addresses:adrian.mehrtashyale.edu(A.B.Mehrtash),mark.hochstrasseryale.edu(M.Hochstrasser).Seminars in Cell and Developmental Biology xxx(xxxx)xxxxxx1084-9521/2018 Published by Elsevi
17、er Ltd.Please cite this article as:Mehrtash,A.B.,Seminars in Cell and Developmental Biology,https:/doi.org/10.1016/j.semcdb.2018.09.013largest class of enzymes in the ubiquitylation cascade,consisting ofapproximately 100 E3s in budding yeast and at least 600 in humans10,11.Consistent with their vast
18、 number,the E3s are the primary UPScomponents responsible for substrate recognition and the remarkablespecificity exhibited by the UPS.Proteins marked for proteasome-mediated degradation typicallycontain at least one poly-ubiquitin chain or multiple mono-ubiquitinadditions 12.Poly-ubiquitin chains a
19、re formed when the C-terminusof a donor ubiquitin is attached to one of the seven lysine residues(K6,K11,K27,K29,K33,K48,and K63)or the alpha-amino group of thefirst methionine(M1)of an acceptor ubiquitin.Ubiquitin modificationsare dynamic and can be removed through the action of deubiquitylatingenz
20、ymes(DUBs)13.Proteins tagged with ubiquitin,most commonlyin K48-linked ubiquitin chains,are recognized by the 26S proteasomeand degraded in an ATP-dependent manner into peptides 14.3.Protein degradation at the ERDue to the massive biosynthetic influx of proteins at the ER,an arrayof factors at the E
21、R promote the proper folding of nascent proteins3,15.Proteins that fail to fold properly or to be correctly modified aredegraded through a branch of the UPS termed ER-Associated De-gradation(ERAD),the process by which proteins are ubiquitylated atthe ER membrane and subsequently degraded.ERAD has an
22、 essentialrole in eliminating proteins that could otherwise prove toxic.Theseprotein quality control substrates include aberrantly folded proteins aswell as protein complex subunits present in excess of their properstoichiometry.Furthermore,ERAD regulates protein levels in responseto cellular demand
23、s 16.In the budding yeast Saccharomyces cerevisiae,there are three ER/NEmembrane-resident E3 ligases responsible for the majority of the pro-tein degradation initiated at these membranes.These include the twocanonical ERAD ligase complexes centered on the Doa10 and Hrd1 E3sas well as the more recent
24、ly characterized Asi complex.While Doa10and Hrd1 have clear homologs in humans,no human orthologs for anyAsi subunits have been defined 17,18.In addition to the mammalianDoa10 ortholog TEB4(MARCH6)and the mammalian Hrd1 orthologsHRD1 and gp78,E3 ligases involved in mammalian ERAD includeTRC8,TMEM129
25、,Rfp2,RNF5/RMA1,RNF145,RNF170,and RNF1851925.There are likely additional ER-resident E3 ligases involved inmammalian ERAD that remain to be characterized 26.The ERADmachinery is capable of degrading a diverse subset of proteins includingthose located within the ER lumen,ER membrane,NE,cytoplasm,andn
26、ucleoplasm.The present review will focus on studies that have illu-minated our understanding of ERAD in yeast and mammals.3.1.ERAD-L and the Hrd1 ubiquitin ligase complexERAD substrates with degradation signals(degrons)located withinthe ER lumen are termed ERAD-L substrates(Fig.1).The degradation of
27、these substrates depends on the UPS machinery,which resides pri-marily within the cytoplasm and nucleoplasm.All the major ERAD E3ligases expose their catalytic RING domains on the cytoplasmic or nu-cleoplasmic side of the ER membrane.Therefore,ERAD-L substratesmust be recognized within the ER lumen
28、and transported across themembrane to undergo ubiquitylation at the ER surface.These substratesmust then be extracted into the cytosol for proteasome-mediated de-gradation.Collectively,protein transport across the ER membrane andsubsequent membrane extraction is termed retrotranslocation and willbe
29、discussed in Section 6.In budding yeast,the Hrd1 E3 ligase complex appears to be the onlyE3 responsible for targeting ERAD-L substrates(Fig.2A).The centralcomponent of the Hrd1 complex is Hrd1,a protein with eight trans-membrane helices(TMs)27 and a catalytic RING domain 28.TheRING interacts with th
30、e E2 enzyme Ubc7 to promote substrate ubi-quitylation.A transmembrane protein,Cue1,helps to tether Ubc7 to theER membrane and is a Ubc7 activator 29.In addition,the Hrd1complex includes the membrane proteins Der1,Hrd3,and Usa1,all ofwhich are involved in the degradation of ERAD-L substrates 30.Hrd3(
31、SEL1L in mammals)is particularly important for the structural in-tegrity of the Hrd1 complex and when absent,Hrd1 undergoes auto-ubiquitylation and rapid degradation 31.Additionally,Hrd3 directlyinteracts with the luminal protein Yos9(OS9 and XTP3-B in mammals).Yos9 is a lectin that recognizes misfo
32、lded glycosylated proteins in theER lumen and delivers them to the Hrd1 complex through its interac-tions with Hrd3 30,32.Misfolded luminal proteins can also be dis-tinguished independently of N-glycan signals and recruited to the Hrd1complex by the luminal Hsp70 chaperone Kar2(BiP or Grp78 inmammal
33、s)32.Usa1(Herp in mammals)promotes the oligomerization of the Hrd1complex and mediates interactions between Der1 and Hrd1 32,33.Der1 is required specifically for the degradation of ERAD-L substrates,yet its exact role remains unclear 32;it may be involved in substraterecognition or the retrotransloc
34、ation of ERAD-L substrates(or both)32,34.Der1 is the founding member of a group of proteins calledDerlins,which are catalytically inactive members of the rhomboidprotease family 35.A Der1 paralog,Dfm1,is also present in yeast.While Dfm1 probably shares a similar topology with Der1 36,37,thetwo yeast
35、 Derlins have distinct roles in ERAD.In contrast to Der1,Dfm1is specifically required for the degradation of Hrd1 membrane sub-strates and probably those of Doa10 as well(see Section 3.2)38.BothDerlins have been implicated in the retrotranslocation process,but fordifferent classes of substrates.The
36、mammalian orthologs of Der1,Derlin-1,-2,and-3,have also been implicated in the retrotranslocationof ERAD substrates(discussed in Section 6)35,39,40.In addition totheir role in retrotranslocation,the Derlins are components of a com-plex,which includes Sec61 and HRD1,involved in degrading specificER p
37、roteins under ER stress conditions in mammalian cells 41,42.In apathwaytermedERstress-inducedpre-emptivequalitycontrol(ERpQC),Derlin-1 facilitates the re-routing of ER-targeted proteinsfrom the translocation pathway to the cytosol for HRD1-mediated de-gradation,thereby reducing the protein folding l
38、oad at the ER 41,42.The number of mammalian ERAD E3 ligases has expanded sig-nificantly relative to yeast.The involvement of the various mammalianE3 ligases in the degradation of luminal proteins is less defined;how-ever,the major components of the ERAD-L machinery in S.cerevisiaeare found in mammal
39、s and ERAD-L substrates are likely recognized andprocessed in a similar manner in yeast and mammals.3.2.The ERAD-M and ERAD-C pathwaysOther than those that are ERAD-L substrates,integral membraneproteins targeted by ERAD are typically categorized into two othersubclasses based on the location of the
40、ir misfolded domains or degrons.Membrane proteins containing a degron within their membrane-span-ning region are termed ERAD-M substrates while those containing adegron in the cytoplasm or nucleoplasm are termed ERAD-C substrates(Fig.1).Following their ubiquitylation,membrane substrates are ret-rotr
41、anslocated into the cytoplasm or nucleoplasm and degraded by theproteasome.The ERAD-C pathway also targets soluble cytoplasmic ornuclear proteins;however,soluble ERAD-C substrates do not requireretrotranslocation for proteasome-mediated degradation.The yeast Hrd1 complex is also involved in degradin
42、g ERAD-Msubstrates.The components of the Hrd1 complex involved in degradingERAD-M substrates have some distinctions from those involved inERAD-L(Fig.2A).The core components of the Hrd1 complex Hrd1,Hrd3 and Usa1 are involved in both ERAD-M and ERAD-L pathways30,33.By contrast,recent data suggest Dfm
43、1 is a component of theERAD-M pathway and not ERAD-L,which instead requires the Der1paralog 38.Yos9,Kar2,and Der1,which have central roles in ERAD-L,are dispensable for ERAD-M,suggesting Hrd1 detects ERAD-M sub-strates by a distinct mechanism.A.B.Mehrtash,M.HochstrasserSeminars in Cell and Developme
44、ntal Biology xxx(xxxx)xxxxxx2Yeast Doa10(degradation of alpha2)was originally identified in ascreen for mutants with defects in the degradation of a soluble nuclearsubstrate bearing the Deg1 degron;Deg1 is a degron from the tran-scription factor MATalpha2 43.While the first Doa10 substrateidentified
45、 was a soluble substrate,numerous membrane substrates wereidentified subsequently 4446.Membrane proteins containing a cy-toplasmic degron(ERAD-C)are primarily targeted for degradation bythe Doa10 complex in S.cerevisiae(Fig.2B).The main component of theDoa10 complex is Doa10,a large E3 ubiquitin lig
46、ase with 14 TMs andan N-terminal RING domain 43.Doa10 is conserved throughout eu-karyotes and the human Doa10 ortholog TEB4/MARCH6 appears tocontain a similar membrane topology and localization 47,48.As noted above,Doa10 functions with two E2s,Ubc6 and Ubc749;both have mammalian orthologs.The Ubc7 o
47、rtholog,Ube2g2,associates with TEB4 to promote K48 chain formation in vitro 50.Ubc6 is a tail-anchored membrane protein that contains two mamma-lian orthologs,Ube2j1 and Ube2j2,which were originally thought notto function with TEB4 51;however,a recent study determined thatUbe2j2 works with TEB4 to d
48、egrade substrates containing the cytosolicCL1 degron,a Doa10-dependent degron in yeast 52,53.Moreover,depletion of Ube2j2 increases protein levels of another TEB4 substrate,squalene monooxygenase(SM),as occurs following proteasomal in-hibition or TEB4 depletion,suggesting Ube2j2 also functions with
49、TEB4to degrade SM.While depletion of the E2 enzyme Ube2D3 was requiredfor complete stabilization of mCherry-CL1 through the TEB4 pathway,Ube2D3 depletion did not significantly affect SM protein levels 52.More work is needed to fully understand which E2s operate with TEB4and how these may differ depe
50、nding on the substrate.Until recently,Doa10 and Hrd1 were thought to be the only ER-resident E3 ligases participating in ERAD in budding yeast;however,recent studies have identified the transmembrane Asi complex as part ofa previously uncharacterized ERAD pathway operating exclusively atthe inner me
