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Protein Targeting and Degradation Are Coupled for Elimination of Mislocalized Proteins

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Protein Targeting and Degradation Are Coupled for Elimination of Mislocalized Proteins LETTER doi:10.1038/nature10181 Protein targeting and degradation are coupled for elimination of mislocalized proteins Tara Hessa1, Ajay Sharma1, Malaiyalam Mariappan1, Heather D. Eshleman1{, Erik Gutierrez1,2 & Ramanujan S. Hegde1{ A substantial proportion of the genome encodes membrane proteins action of puromycin (Fig. 1c and Supplementary Fig. 5). An unrelated that are delivered to the endoplasmic reticulum by dedicated target- membrane protein behaved similarly (Supplementary Fig. 6). ing pathways1. Membrane proteins that fail targeting must be rapidly Efficient ubiquitination of PrP was strongly dependent on unpro- degraded to avoid aggregation and disruption of cytosolic protein cessed hydrophobic signals at the amino and carboxy termini homeostasis2,3. The mechanisms of mislocalized protein (MLP) (Fig. 1d). Conversely, green fluorescent protein (GFP) became a sub- degradation are unknown. Here we reconstitute MLP degradation strate for ubiquitination when hydrophobic targeting signals were added in vitro to identify factors involved in this pathway. We find that (Supplementary Fig. 4). Ubiquitination was therefore not solely a con- nascent membrane proteins tethered to ribosomes are not sub- sequence of protein misfolding,because PrP lackingboth the N-terminal strates for ubiquitination unless they are released into the cytosol. targeting signal (denoted DSS) and the C-terminal GPI-anchoring signal Their inappropriate release results in capture by the Bag6 com- (DGPI) was misfolded owing to its lack of glycosylation and disulphide plex, a recently identified ribosome-associating chaperone4.Bag6- bond formation, but was poorly ubiquitinated. This finding suggested complex-mediated capture depends on the presence of unprocessed the existence of a specialized pathway for hydrophobic-domain- or non-inserted hydrophobic domains that distinguish MLPs from containing MLPs that works more rapidly than traditional quality con- potential cytosolic proteins. A subset of these Bag6 complex ‘clients’ trol pathways, which engage only after repeated failures at folding15,16. are transferred to TRC40 for insertion into the membrane, whereas To identify factors involved in the MLP degradation pathway, we the remainder are rapidly ubiquitinated. Depletion of the Bag6 com- combined biochemical fractionation and functional reconstitution plex selectively impairs the efficient ubiquitination of MLPs. Thus, assays. We produced a translation-competent fractionated RRL (Fr- by its presence on ribosomes that are synthesizing nascent mem- RRL) (Supplementary Fig. 7) that selectively decreased the ubiquitina- brane proteins, the Bag6 complex links targeting and ubiquitination tion of non-translocated PrP (Fig. 2a) and other MLPs (Supplementary pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic Time folding machinery. a b RM: –+ –+ (min) 369 201512 25 504030 60 Protein targeting and translocation to the endoplasmic reticulum 5,6 (ER) are not perfectly efficient , thereby necessitating pathways for Ub Ub Ubiq the degradation of MLPs that have been inappropriately released into the cytosol. For example, mammalian prion protein (PrP), a widely Glyc Pre expressed glycosyl phosphatidylinositol (GPI)-anchored cell surface Pro Total glycoprotein, displays ,5–15% translocation failure in vitro and in Total Ubiq vivo2,3,5–10. This non-translocated population of PrP is degraded effi- c d ciently by a proteasome-dependent pathway, limiting the cytosolic PrP Term Δ Δ Δ Δ Δ Δ 2,3,9,10 Trunc Wild typeSS SSPrl-SSGPINPY-SSWild typeSS SSPrl-SSGPINPY-SS levels at steady state . Prompt degradation is essential because RNase:– –+ Cyt:–+ –+ mislocalized PrP can aggregate, make inappropriate interactions, 2,11–14 Ub and cause cell death and neurodegeneration . The pathways for Ub Ub efficient disposal of MLPs, however, are not known. To study this problem, we reconstituted the ubiquitination of mis- Pre localized PrP in vitro. Radiolabelled PrP synthesized in rabbit reticulo- Total Ubiq cyte lysate (RRL) supplemented with ER-derived rough microsomes Puromycin Total Ubiq was predominantly translocated into the ER, processed and glycosy- Figure 1 | Non-translocated PrP is rapidly ubiquitinated. a, The translation lated (Fig. 1a). However, various conditions that reduced the extent of of radiolabelled PrP in RRL, with or without rough microsomes (RMs), was translocation—such as omission of rough microsomes, inactivation of analysed directly (left) or after isolation of ubiquitinated (ubiq) products (right) signal recognition particle (SRP)-dependent targeting or blocking of by using SDS–PAGE and autoradiography. Glycosylated (glyc), precursor translocation through the translocon—all resulted in increased PrP (pre), processed (pro) and ubiquitinated (Ub) bands are indicated. b, Time ubiquitination in a lysine-dependent manner (Fig. 1a and Supplemen- course of PrP synthesis (bottom) and PrP ubiquitination (top) in vitro. c,PrP tary Figs 1–3). Other mislocalized secretory and membrane proteins containing a termination codon (term) or lacking this codon (trunc) was were also similarly ubiquitinated in the cytosol (Supplementary Fig. 4). translated in vitro. Truncated PrP was released using puromycin, in the absence or presence of cytosol (cyt), and total protein and ubiquitination were analysed. The ubiquitination of mislocalized PrP closely parallels PrP synthesis The arrowhead indicates tRNA-containing PrP, which can be digested by (Fig. 1b), suggesting that ubiquitination is rapid. Yet, ubiquitination RNase. d, Wild-type PrP or constructs lacking the signal sequence (DSS) or occurred strictly post-translationally, because full-length PrP that was both the signal sequence and GPI anchor (DSSDGPI) were analysed directly or tethered as a nascent peptidyl-transfer RNA to the ribosome was not after isolation of ubiquitinated products. Prl-SS and NYP-SS contain signal ubiquitinated until it had been released into the cytosol through the sequence from preprolactin and neuropeptide Y, respectively. 1Cell Biology and Metabolism Program, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. 2Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA. {Present addresses: Department of Physiology, University of California, San Francisco, California 94158, USA (H.D.E.); MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK (R.S.H.). 00 MONTH 2011 | VOL 000 | NATURE | 1 ©2011 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER Fig. 8) but not ubiquitination in general (Supplementary Fig. 7). The (Supplementary Figs 14 and 15, and data not shown). Thus, the Bag6 missing factor in Fr-RRL (other than ubiquitin, which we included in complex binds to multiple MLPs through their hydrophobic domains all assays) proved to be the E2 ubiquitin-conjugating enzyme UBCH5 and has a broader specificity than only binding tail-anchored proteins. (also known as UBE2D1) (Fig. 2b and Supplementary Figs 8 and 9). To determine when the Bag6 complex first captures MLPs, we ana- Because UBCH5 restored ubiquitination equally well when added dur- lysed ribosome-nascent chains (RNCs) of membrane proteins. When a ing or after PrP translation (Fig. 2b), we surmised that at least a certain transmembrane domain (TMD) emerged from the ribosomal ‘tunnel’, population of PrP remains in a ubiquitination-competent state. a direct interaction with SRP54 (the signal-sequence-binding subunit Indeed, PrP and other MLPs that were affinity purified from Fr-RRL of the SRP) was detected by crosslinking experiments (Fig. 3a–c). By under native conditions could be ubiquitinated simply by adding puri- contrast, the Bag6 complex, even though it has been found to reside on fied E1, UBCH5, ubiquitin and ATP (Fig. 2c and Supplementary Fig. 10). such RNCs and is abundant in the cytosol4, did not make direct contact To identify factors that maintain the ubiquitination competence of with the substrate (Fig. 3b, c). When the TMD was still inside the MLPs, the Fr-RRL translation products were separated by size in a ribosomal tunnel, the RNC was not crosslinked to either BAG6 or sucrose gradient, and each fraction was subjected to parallel ubiquiti- SRP54 (Fig. 3c), even though both complexes can be recruited to such nation and chemical crosslinking analyses (Fig. 2d and Supplementary ribosomes4,18. After puromycin release of each of these RNCs (with the Fig. 11). The fractions retaining maximum ubiquitination competence TMD inside versus outside the ribosomal tunnel), BAG6 crosslinking for two different substrates correlated well with a ,150-kDa cross- was observed (Fig. 3b, c). Thus, the Bag6 complex captures substrates linking partner (Fig. 2d and Supplementary Fig. 11). This interaction concomitant with or after the release of nascent chains from the ribo- was direct (Supplementary Fig. 12) and was strongly dependent on the some; these same hydrophobic domains are bound by the SRP as long presence of unprocessed N- and C-terminal signals in PrP (Fig. 2e and as the TMD is exposed as an RNC19. Supplementary Fig. 13), correlating with the requirements for ubiqui- Earlier analysis of tail-anchored and non-tail-anchored membrane tination (Fig. 1d). On the basis of molecular weight, dependence on proteins had shown that only tail-anchoredmembraneproteinsareeffi- hydrophobic domains for interaction and migration position in the ciently loaded
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