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Routing Misfolded Proteins through the Multivesicular Body (MVB) Pathway Protects against Proteotoxicity*

  • Songyu Wang
    Affiliations
    Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore

    Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore
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  • Guillaume Thibault
    Affiliations
    Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore
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  • Davis T.W. Ng
    Correspondence
    To whom correspondence should be addressed: Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore. Fax: 65-6872-7007
    Affiliations
    Temasek Life Sciences Laboratory, National University of Singapore, Singapore 117604, Singapore

    Department of Biological Sciences, National University of Singapore, Singapore 117604, Singapore
    Search for articles by this author
  • Author Footnotes
    * This work was supported by funds from the Temasek Trust and by grants from the Singapore Millennium Foundation (predoctoral fellowship (to S. W.) and postdoctoral fellowship (to G. T.)).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and Figs. S1–S8.
Open AccessPublished:June 27, 2011DOI:https://doi.org/10.1074/jbc.M111.233346
      The secretory pathway maintains multiple quality control checkpoints. Initially, endoplasmic reticulum-associated degradation pathways monitor protein folding to retain and eliminate aberrant products. Despite its broad client range, some molecules escape detection and traffic to Golgi membranes. There, a poorly understood mechanism termed Golgi quality control routes aberrant proteins for lysosomal/vacuolar degradation. To better understand Golgi quality control, we examined the processing of the obligate substrate Wsc1p. Misfolded Wsc1p does not use routes of typical vacuolar membrane proteins. Instead, it partitions into intralumenal vesicles of the multivesicular body (MVB) pathway, mediated by the E3 ubiquitin ligase Rsp5p. Its subsequent transport to the vacuolar lumen is essential for complete molecule breakdown. Surprisingly, the transport mode plays a second crucial function in neutralizing potential substrate toxicity. Eliminating the MVB sorting signal diverted molecules to the vacuolar limiting membrane, resulting in the generation of toxic by-products. These data demonstrate a new role of the MVB pathway in protein quality control.

      Introduction

      Protein quality control mechanisms ensure the fidelity of the proteome by partitioning polypeptides based on conformational states. Correctly folded proteins can proceed to and remain at their sites of function, whereas aberrant molecules are slated for elimination. In the early secretory pathway, endoplasmic reticulum-associated degradation (ERAD)
      The abbreviations used are: ERAD
      endoplasmic reticulum-associated degradation
      ER
      endoplasmic reticulum
      GQC
      Golgi quality control
      MVB
      multivesicular body
      CPY
      carboxypeptidase Y
      PGK
      3-phosphoglyceric phosphokinase
      DIC
      differential interference contrast
      ESCRT
      endosomal sorting complexes required for transport.
      and autophagic pathways specialize in seeking misfolded polypeptides and mediate their degradation (for a review, see Ref.
      • Vembar S.S.
      • Brodsky J.L.
      ). Although these mechanisms seemed sufficiently comprehensive, reports of aberrant molecules trafficking out of the ER undetected suggest that the “molecular sieve” is somewhat porous (
      • Spear E.D.
      • Ng D.T.
      ,
      • Ashok A.
      • Hegde R.S.
      ,
      • Apaja P.M.
      • Xu H.
      • Lukacs G.L.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ). Even a minor flaw could be disastrous because misfolded proteins are often toxic if allowed to accumulate. This scenario is averted because analyses of ERAD-independent substrates revealed post-ER quality control mechanisms that efficiently capture the wayward molecules. Some molecules are caught in the Golgi apparatus, whereas others continue on to the plasma membrane. At both sites, specialized mechanisms sort and transport aberrant proteins to the lysosomes and vacuoles (lysosome-like organelles in fungi) for degradation.
      The surveillance mechanism at the Golgi apparatus is termed Golgi quality control (GQC) (for reviews, see Refs.
      • Arvan P.
      • Zhao X.
      • Ramos-Castaneda J.
      • Chang A.
      ,
      • Anelli T.
      • Sitia R.
      ,
      • Hegde R.S.
      • Ploegh H.L.
      ). Like ERAD, it recognizes a variety of aberrant soluble and membrane proteins (
      • Spear E.D.
      • Ng D.T.
      ,
      • Ashok A.
      • Hegde R.S.
      ,
      • Chang A.
      • Fink G.R.
      ,
      • Hong E.
      • Davidson A.R.
      • Kaiser C.A.
      ,
      • Jenness D.D.
      • Li Y.
      • Tipper C.
      • Spatrick P.
      ,
      • Coughlan C.M.
      • Walker J.L.
      • Cochran J.C.
      • Wittrup K.D.
      • Brodsky J.L.
      ). To be effective, its stringency must be greater than ERAD. This is apparently the case because GQC efficiently ensnares escaped ERAD substrates (
      • Spear E.D.
      • Ng D.T.
      ,
      • Kincaid M.M.
      • Cooper A.A.
      ) and recognizes a form of bovine pancreatic trypsin inhibitor with increased structural flexibility but not grossly misfolded (
      • Coughlan C.M.
      • Walker J.L.
      • Cochran J.C.
      • Wittrup K.D.
      • Brodsky J.L.
      ). The plasma membrane mechanism, called peripheral quality control, is dedicated to the surveillance of membrane proteins (
      • Apaja P.M.
      • Xu H.
      • Lukacs G.L.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ). This mechanism is distinct from the clearance of folded receptor proteins in the early steps due to its reliance on the Hsp70/Hsp90 molecular chaperone systems (
      • Apaja P.M.
      • Xu H.
      • Lukacs G.L.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ). This is notable because the same chaperone classes are employed for the quality control of ER and cytosolic proteins (for a review, see Ref.
      • Buchberger A.
      • Bukau B.
      • Sommer T.
      ). Substrate ubiquitination is used to signal endocytosis and sorting into the multivesicular body (MVB) endosomal pathway for degradation in lysosomes (for a review, see Ref.
      • Hicke L.
      • Dunn R.
      ).
      The transport route for GQC substrates from the Golgi is not known. In budding yeast, proteins traffic to the vacuole using one of two general routes. The first involves trafficking proteins in Golgi-derived vesicles to an endosomal prevacuolar compartment. This pathway was defined genetically in part by the class E vps (vacuolar protein sorting) mutants (
      • Raymond C.K.
      • Howald-Stevenson I.
      • Vater C.A.
      • Stevens T.H.
      ,
      • Piper R.C.
      • Cooper A.A.
      • Yang H.
      • Stevens T.H.
      ,
      • Rieder S.E.
      • Banta L.M.
      • Köhrer K.
      • McCaffery J.M.
      • Emr S.D.
      ). Class E mutants cause formation of exaggerated prevacuolar compartments containing soluble and membrane vacuolar hydrolases, unrecycled Golgi proteins, and endocytosed proteins (
      • Raymond C.K.
      • Howald-Stevenson I.
      • Vater C.A.
      • Stevens T.H.
      ,
      • Piper R.C.
      • Cooper A.A.
      • Yang H.
      • Stevens T.H.
      ,
      • Babst M.
      • Sato T.K.
      • Banta L.M.
      • Emr S.D.
      ,
      • Piper R.C.
      • Bryant N.J.
      • Stevens T.H.
      ,
      • Odorizzi G.
      • Babst M.
      • Emr S.D.
      ). Thus, the prevacuolar compartment represents a major trafficking hub for vacuolar/lysosomal transport that also includes the MVB pathway. This pathway is termed the “CPY pathway” because carboxypeptidase Y is a well studied client. A distinct vacuolar transport pathway was defined by the membrane protein alkaline phosphatase (
      • Piper R.C.
      • Bryant N.J.
      • Stevens T.H.
      ,
      • Cowles C.R.
      • Snyder W.B.
      • Burd C.G.
      • Emr S.D.
      ,
      • Stepp J.D.
      • Huang K.
      • Lemmon S.K.
      ). The alkaline phosphatase pathway is not as well understood as the CPY pathway, but there are clear differences. Whereas the alkaline phosphatase pathway is known only to deliver membrane-bound cargo to vacuolar limiting membrane, the CPY pathway can do that and also places membrane proteins entirely into the lumen using the MVB pathway. For membrane-integrated GQC substrates, there is no obvious restriction for the route used. The MVB pathway provides a good mechanism because molecules are transported entirely into the vacuolar lumen on intralumenal vesicles. By contrast, the alternative alkaline phosphatase pathway results in membrane protein insertion into vacuolar limiting membrane. Although this might present a topological problem for degradation, the invagination and hydrolysis of vacuolar membranes and their contents through microautophagy would overcome the limitation if coupled to protein quality control (
      • Kunz J.B.
      • Schwarz H.
      • Mayer A.
      ,
      • Dubouloz F.
      • Deloche O.
      • Wanke V.
      • Cameroni E.
      • De Virgilio C.
      ).
      We previously reported that a plasma membrane protein, Wsc1p, is an endogenous, obligate substrate of post-ER quality control (
      • Wang S.
      • Ng D.T.
      ). Wsc1p is a single-spanning integral membrane protein that functions as a sensor for cell wall integrity in budding yeast (
      • Verna J.
      • Lodder A.
      • Lee K.
      • Vagts A.
      • Ballester R.
      ,
      • Jacoby J.J.
      • Nilius S.M.
      • Heinisch J.J.
      ). Mutations disrupting the luminal folding of Wsc1p are not detected by ERAD, in part due to the inability of the ERAD chaperone Kar2p/BiP to recognize the variants (
      • Wang S.
      • Ng D.T.
      ). Instead, the Wsc1p cytoplasmic domain contains a dominant ER export signal that directs its transport regardless of the luminal folding state. For Wsc1p quality control, sorting occurs in the Golgi apparatus. Folded Wsc1p progresses to the plasma membrane, whereas misfolded molecules partition to the vacuole, dependent on the Vps10p cargo-sorting factor (
      • Wang S.
      • Ng D.T.
      ). These characteristics make Wsc1p an ideal model to uncover the mechanisms underlying GQC. Although both soluble and membrane GQC substrates can utilize Vps10p, the subsequent steps of transport are unknown (
      • Hong E.
      • Davidson A.R.
      • Kaiser C.A.
      ,
      • Jørgensen M.U.
      • Emr S.D.
      • Winther J.R.
      ). In this study, Wsc1p variants were used to uncover the sorting and transport mechanism from the Golgi apparatus. We show that GQC and peripheral quality control pathways converge at the MVB endosomal sorting compartment. Importantly, we demonstrate that the use of the MVB pathway ensures whole molecule degradation and prevents toxic degradation products that would otherwise form through other vacuolar transport routes.

      DISCUSSION

      The range of protein quality control mechanisms is far wider than previously thought. The breadth is not surprising, given their importance in maintaining cellular protein homeostasis. Although early studies focused on those stationed at sites of de novo protein synthesis, there is now great interest in less understood pathways that can be found throughout the cell (
      • Ashok A.
      • Hegde R.S.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ,
      • Chang A.
      • Fink G.R.
      ,
      • Hong E.
      • Davidson A.R.
      • Kaiser C.A.
      ,
      • Prasad R.
      • Kawaguchi S.
      • Ng D.T.
      ,
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ,
      • Kaganovich D.
      • Kopito R.
      • Frydman J.
      ,
      • Heck J.W.
      • Cheung S.K.
      • Hampton R.Y.
      ,
      • Heo J.M.
      • Livnat-Levanon N.
      • Taylor E.B.
      • Jones K.T.
      • Dephoure N.
      • Ring J.
      • Xie J.
      • Brodsky J.L.
      • Madeo F.
      • Gygi S.P.
      • Ashrafi K.
      • Glickman M.H.
      • Rutter J.
      ). Instead of just monitoring the folding of polypeptides as they are made, these mechanisms also police populations of functional proteins for those going bad. Thus, strategies suited only for actively folding proteins, like the mannose timer hypothesis in ERAD (
      • Jakob C.A.
      • Bodmer D.
      • Spirig U.
      • Battig P.
      • Marcil A.
      • Dignard D.
      • Bergeron J.J.
      • Thomas D.Y.
      • Aebi M.
      ,
      • Quan E.M.
      • Kamiya Y.
      • Kamiya D.
      • Denic V.
      • Weibezahn J.
      • Kato K.
      • Weissman J.S.
      ,
      • Clerc S.
      • Hirsch C.
      • Oggier D.M.
      • Deprez P.
      • Jakob C.
      • Sommer T.
      • Aebi M.
      ), would not apply there.
      The peripheral quality control system appears to be charged with both roles. For example, the multispanning protein Pma1p is subject to ERAD if it is grossly misfolded by mutation. One allele, pma1-7, is caught by GQC and diverted to the vacuole for turnover (
      • Chang A.
      • Fink G.R.
      ). Other mutants, undetected by either pathway, traffic to the plasma membrane, where they rapidly endocytose and traffic to the vacuole (
      • Gong X.
      • Chang A.
      ,
      • Liu Y.
      • Sitaraman S.
      • Chang A.
      ). The reasons for these sorting patterns are unclear because specific features, recognized by one system and not others, are not known. The plasma membrane system can also detect post-maturation conformational changes. In mammalian cells, the most common disease allele of the cystic fibrosis transconductance regulator is ΔF508 (
      • Cheng S.H.
      • Gregory R.J.
      • Marshall J.
      • Paul S.
      • Souza D.W.
      • White G.A.
      • O'Riordan C.R.
      • Smith A.E.
      ). At 37 °C, ΔF508 is retained in the ER and degraded by ERAD. However, shifting cells to 26 °C allows the maturation and trafficking of the mutant to the plasma membrane, where it is at least partially functional (
      • Denning G.M.
      • Anderson M.P.
      • Amara J.F.
      • Marshall J.
      • Smith A.E.
      • Welsh M.J.
      ,
      • Lukacs G.L.
      • Mohamed A.
      • Kartner N.
      • Chang X.B.
      • Riordan J.R.
      • Grinstein S.
      ,
      • Jensen T.J.
      • Loo M.A.
      • Pind S.
      • Williams D.B.
      • Goldberg A.L.
      • Riordan J.R.
      ,
      • Ward C.L.
      • Omura S.
      • Kopito R.R.
      ). Because of this effect, getting mutant forms of cystic fibrosis transconductance regulator to bypass the ERAD checkpoint has been a major research goal as a therapeutic strategy. However, the existence of a plasma membrane quality control mechanism complicates the strategy. Returning cells to 37 °C, and presumably the corresponding conformation, triggers ΔF508 recognition, endocytosis, and transport to lysosomes by the MVB pathway (
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ,
      • Glozman R.
      • Okiyoneda T.
      • Mulvihill C.M.
      • Rini J.M.
      • Barriere H.
      • Lukacs G.L.
      ,
      • Sharma M.
      • Pampinella F.
      • Nemes C.
      • Benharouga M.
      • So J.
      • Du K.
      • Bache K.G.
      • Papsin B.
      • Zerangue N.
      • Stenmark H.
      • Lukacs G.L.
      ). Thus, the importance of understanding these mechanisms cannot be overstated. Lukacs and co-workers (
      • Apaja P.M.
      • Xu H.
      • Lukacs G.L.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ) recently reported the role of cytosolic chaperones in recognizing misfolded proteins at the plasma membrane. Interestingly, some of these are known components of other quality control systems suggesting mechanistic overlaps. These studies demonstrate the necessity to account for peripheral quality control before the therapeutic strategy can be viable.
      In this study, we report that the GQC substrate Wsc1* sorts to the MVB pathway for turnover (Fig. 8A), making it a point of convergence between cell surface and Golgi mechanisms. Unlike plasma membrane receptors, which tend to recycle if MVB sorting is subverted, misfolded Wsc1p traffics to the vacuole even if the pathway cannot be utilized. This suggests that the signals required for vacuolar transport and MVB sorting are separable. This made it possible to demonstrate that the route is essential for complete substrate degradation. Diversion of misfolded Wsc1p to the alternative vacuolar transport route caused the accumulation of partially degraded molecules still embedded in the vacuolar membrane (Fig. 8B). This result provided evidence that the normal transport pathways used by resident vacuolar membrane proteins are not suitable for protein quality control. Remarkably, we discovered that the resulting “luminally sheared” form of Wsc1p is highly toxic. This form disrupts vacuolar membrane integrity and might indirectly affect lipid homeostasis. This showed that the efficient whole-molecule degradation provided by the MVB pathway could also provide a protective function for the cell (Fig. 8). Taken together, these data provide a physiological basis for trafficking misfolded proteins through the MVB pathway. Because peripheral quality control substrates ultimately use the same pathway, the principles gleaned from the Wsc1* studies are likely to be more generally applicable.
      Figure thumbnail gr8
      FIGURE 8Model of the MVB-dependent pathway for the transport of misfolded Wsc1p. A, normally, misfolded Wsc1p exits the ER and transits through the Golgi apparatus. It is next sorted to the MVB pathway and degraded in its entirety within the vacuolar lumen. This mechanism requires the ubiquitin ligase Rsp5p and ubiquitination of the Wsc1p cytoplasmic domain. B, misfolded GQC substrates missorted to the vacuolar limiting membrane. Degradation proceeds but is constrained by the membrane, leading to partial degradation products still integrated in the membrane. These aberrant products disrupt vacuolar membranes, which can lead to cell death.
      With the MVB pathway being a convergence point, why are two (or more) post-ER surveillance sites needed? The studies from the Lukacs and Gardner laboratories (
      • Apaja P.M.
      • Xu H.
      • Lukacs G.L.
      ,
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ,
      • Gardner R.G.
      • Nelson Z.W.
      • Gottschling D.E.
      ,
      • Sharma M.
      • Pampinella F.
      • Nemes C.
      • Benharouga M.
      • So J.
      • Du K.
      • Bache K.G.
      • Papsin B.
      • Zerangue N.
      • Stenmark H.
      • Lukacs G.L.
      ) may have already provided the answer. In both cases, the groups examined the fate of proteins where folding states can be controlled by temperature. At low temperatures, the proteins are folded and functional. After shifting to restrictive temperatures, the mutant proteins change conformations to sufficiently pique the attention of local quality control mechanisms at the plasma membrane and nucleus. These studies suggest that quality control mechanisms are at play anywhere there are proteins, folded or unfolded. Thus, the other role of GQC may be to continuously monitor the integrity of folded resident Golgi proteins. The discovery of its role in capturing misfolded proteins undetected by ERAD could be explained simply by the fact that such model molecules were easier to generate. However, there are also clear differences between plasma membrane and Golgi systems that might impact their client range. In GQC, misfolded luminal domains like those in Wsc1* are detected by Golgi cargo-sorting factors, such as Vps10p (soluble substrates are entirely luminal) (
      • Hong E.
      • Davidson A.R.
      • Kaiser C.A.
      ,
      • Wang S.
      • Ng D.T.
      ,
      • Jørgensen M.U.
      • Emr S.D.
      • Winther J.R.
      ). By contrast, misfolded soluble proteins that can make it to the plasma membrane are secreted, suggesting the absence of a surveillance mechanism on the exterior of the cell, which is the topologic equivalent of the organelle lumen (
      • Hong E.
      • Davidson A.R.
      • Kaiser C.A.
      ,
      • Nakatsukasa K.
      • Okada S.
      • Umebayashi K.
      • Fukuda R.
      • Nishikawa S.
      • Endo T.
      ). Indeed, the ΔF508 lesion of cystic fibrosis transconductance regulator affects the conformation of its cytoplasmic domain, and the known recognition factors are cytosolic (
      • Okiyoneda T.
      • Barrière H.
      • Bagdány M.
      • Rabeh W.M.
      • Du K.
      • Höhfeld J
      • Young J.C.
      • Lukacs G.L.
      ). Although it is tempting to suggest a division of labor between these pathways, the mutations in the pma1-7 allele that make it a substrate of GCQ probably cause conformational changes to its cytosolic domains, suggesting that GQC is not restricted to luminal abnormalities (
      • Chang A.
      • Fink G.R.
      ).
      In this paper, we report the transport mechanism used by GCQ for the obligate substrate Wsc1p. Major questions remain. The most immediate is how misfolded proteins are detected in the Golgi lumen. Although Vps10p is an important factor, how it and other yet to be identified factors differentiate folded and unfolded proteins remains mysterious. Another question is the molecular mechanism of toxicity for Wsc1*-6R degradation by-products. Although the severity of their effects on vacuolar membrane may be sufficient to explain their toxicity, it remains to be determined how it manifests simply through the degradation of the luminal domain. To our knowledge, this is the first instance in which such a phenomenon has been observed in vivo. Although many questions remain, recent studies demonstrate that cellular protein homeostasis relies on a complex network of quality control systems, some of which remain to be discovered.

      Acknowledgments

      We thank Dr. Chris Kaiser for providing the rsp5-1 strain. We are grateful to the TLL/NUS core facilities, especially Dr. Graham Wright and Dr. Meredith Calvert for providing excellent technical support for imaging studies and Dr. Xuezhi Ouyang for transmission electron microscopy sample preparation. We also acknowledge Chengchao Xu and Alisha Chakrabarti for critical reading of the manuscript.

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