MARCH6 and TRC8 facilitate the quality control of cytosolic and tail‐anchored proteins
2018; Springer Nature; Volume: 19; Issue: 5 Linguagem: Inglês
10.15252/embr.201745603
ISSN1469-3178
AutoresSandra Stefanovic‐Barrett, Anna S. Dickson, Stephen P. Burr, James C. Williamson, Ian Lobb, Dick J. H. van den Boomen, Paul J. Lehner, James A. Nathan,
Tópico(s)Transgenic Plants and Applications
ResumoArticle8 March 2018Open Access Transparent process MARCH6 and TRC8 facilitate the quality control of cytosolic and tail-anchored proteins Sandra Stefanovic-Barrett Sandra Stefanovic-Barrett Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Anna S Dickson Anna S Dickson Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Stephen P Burr Stephen P Burr Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author James C Williamson James C Williamson Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Ian T Lobb Ian T Lobb Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Dick JH van den Boomen Dick JH van den Boomen Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Paul J Lehner Paul J Lehner Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author James A Nathan Corresponding Author James A Nathan [email protected] orcid.org/0000-0002-0248-1632 Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Sandra Stefanovic-Barrett Sandra Stefanovic-Barrett Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Anna S Dickson Anna S Dickson Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Stephen P Burr Stephen P Burr Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author James C Williamson James C Williamson Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Ian T Lobb Ian T Lobb Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Dick JH van den Boomen Dick JH van den Boomen Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Paul J Lehner Paul J Lehner Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author James A Nathan Corresponding Author James A Nathan [email protected] orcid.org/0000-0002-0248-1632 Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK Search for more papers by this author Author Information Sandra Stefanovic-Barrett1, Anna S Dickson1, Stephen P Burr1, James C Williamson1, Ian T Lobb1, Dick JH Boomen1, Paul J Lehner1 and James A Nathan *,1 1Department of Medicine, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK *Corresponding author. Tel: +44 1223 748483; E-mail: [email protected] EMBO Reports (2018)19:e45603https://doi.org/10.15252/embr.201745603 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Misfolded or damaged proteins are typically targeted for destruction by proteasome-mediated degradation, but the mammalian ubiquitin machinery involved is incompletely understood. Here, using forward genetic screens in human cells, we find that the proteasome-mediated degradation of the soluble misfolded reporter, mCherry-CL1, involves two ER-resident E3 ligases, MARCH6 and TRC8. mCherry-CL1 degradation is routed via the ER membrane and dependent on the hydrophobicity of the substrate, with complete stabilisation only observed in double knockout MARCH6/TRC8 cells. To identify a more physiological correlate, we used quantitative mass spectrometry and found that TRC8 and MARCH6 depletion altered the turnover of the tail-anchored protein heme oxygenase-1 (HO-1). These E3 ligases associate with the intramembrane cleaving signal peptide peptidase (SPP) and facilitate the degradation of HO-1 following intramembrane proteolysis. Our results highlight how ER-resident ligases may target the same substrates, but work independently of each other, to optimise the protein quality control of selected soluble and tail-anchored proteins. Synopsis Using near-haploid and CRISPR-Cas9 mutagenesis screens, this study identifies overlapping functions for the ER-resident E3 ligases, MARCH6 and TRC8, in the quality control of selected soluble and tail-anchored proteins at the cytosolic face of the ER. Protein quality control of soluble misfolded proteins, such as the CL1 degron, is dependent on their association with the ER membrane. The ER-resident E3 ligases, MARCH6 and TRC8, both facilitate the degradation of mCherry-CL1, but work independently of each other. Quantitative mass spectrometry identifies the tail-anchored protein, HO-1, as a substrate of both MARCH6 and TRC8. MARCH6 and TRC8 associate with signal peptide peptidase to facilitate the degradation and protein quality control of HO-1 following intramembrane proteolysis. Introduction Intracellular protein quality control is essential to remove misfolded proteins that may otherwise promote aggregate formation and toxicity, leading to conditions such as Alzheimer's disease, Parkinson's disease and cystic fibrosis 1, 2. Terminally misfolded proteins typically have exposed hydrophobic domains prone to aggregation 3 and can be ubiquitinated and selectively targeted for proteasomal degradation. In yeast, these proteins are ubiquitinated by quality control E3 ligases, including the cytosolic Ubr1p and San1p ligases 4-6 and the endoplasmic reticulum (ER)-resident ligase Doa10p 7-9. The mammalian ubiquitin machinery required to remove aberrantly folded proteins is less clearly defined. The increased diversity and complexity seen in mammalian enzymes involved in protein quality control are in part driven by an expansion of E2 and E3 ligases 10, highlighting the need to elucidate their detailed functions. ER-associated degradation (ERAD) provides the major mechanism for protein quality control at the ER membrane, facilitating the dislocation of proteins from the ER for degradation by the proteasome within the cytosol. In yeast, three E3 ligases promote ERAD: Hrd1p and Doa10p recognise their substrates at the ER 11, while the Asi E3 ligase complex facilitates ERAD at the inner nuclear membrane 12, 13. In mammalian cells, orthologues of Hrd1p (HRD1) and Doa10p (MARCH6, also known as TEB4) support ERAD 14-17, but there has also been a marked expansion of ER-resident E3 ligases. Some of these are homologous to Hrd1p (e.g. gp78) 18, while others do not resemble the yeast ligases, such as the RING-H2 containing TRC8 19 and TMEM129 20, 21, both implicated in the human cytomegalovirus-mediated ERAD of MHC class I molecules. Several reporter systems for investigating protein quality control have been developed, including the generation of artificial substrates with exposed hydrophobic domains typical of misfolded proteins 22. The CL1 degron was originally identified in a Saccharomyces cerevisiae (S. cerevisiae) screen for proteasome substrates as an unstable C-terminal extension that promotes rapid degradation 7, 23, and has been widely used to report on proteasome activity in the context of neurological diseases associated with cytosolic protein aggregation 24, 25. In yeast, attachment of CL1 to cytosolic proteins (e.g. Ura3p) leads to their degradation via the ERAD degradation machinery, including the ER-resident E3 ligase Doa10p 23. In mammalian cells, fusion of this 16 amino acid amphipathic helix to usually stable proteins also leads to their proteasomal destruction 22, 26, but the molecular machinery required for degradation is not known. Here, we use the CL1 degron as a model substrate in unbiased mutagenesis screens to identify the ubiquitin machinery required for degrading misfolded soluble proteins in human cells. We show that two ER-resident ubiquitin ligases, TRC8 and MARCH6, facilitate the proteasomal degradation of the soluble fluorescent CL1 substrate (mCherry-CL1) at the cytosolic face of the ER membrane. To establish the biological significance of this pathway, we use a quantitative proteomic approach to identify potential substrates of these ER-resident ligases. We find that selected tail-anchored proteins that undergo intramembrane proteolysis by signal peptide peptidase (SPP) 27 share some properties with the mCherry-CL1 degron, in that their degradation can be mediated by both TRC8 and MARCH6. Results A near-haploid genetic screen identifies TRC8 and MARCH6 as ubiquitin E3 ligases involved in degradation of the CL1 degron We established a human mutagenesis screen in near-haploid KBM7 cells (karyotype 25, XY, +8, Ph+) 28 to identify genes required for protein quality control, using a fluorescent CL1 degron as an unstable hydrophobic proteasome substrate. This screening methodology provides a powerful unbiased forward genetic approach, which has led to the identification of novel pathogen restriction factors, epigenetic regulators, ERAD components and genes involved in regulation of the hypoxia response 28-33. We designed our fluorescent reporter to encode the CL1 amphipathic sequence as a C-terminal extension of mCherry (Fig 1A and B). KBM7 and HeLa cells stably expressing this reporter showed low mCherry-CL1 fluorescence and protein levels, which increased following incubation with the proteasome inhibitor, bortezomib (Velcade), consistent with constitutive proteasome-mediated degradation of the reporter (Fig 1C and D). Confocal immunofluorescence demonstrated that mCherry-CL1 was diffusely distributed within the cytosol following proteasome inhibition (Fig 1E), as observed by others for a GFP-CL1 fusion protein in human cells 22, 26. Figure 1. A near-haploid genetic screen identifies genes required for cytosolic protein quality control in human cells A. Schematic of the mCherry-CL1 reporter. B. Diagram of the 16 amino acid CL1 amphipathic helix using a helical wheel prediction (http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). C, D. Clonal populations of KBM7 (left) or HeLa cells (right) expressing the mCherry-CL1 reporter were incubated with 40 nM (KBM7) or 100 nM (HeLa) bortezomib (Btz) for 6 h and mCherry levels measured by flow cytometry (C) or immunoblot (D). *Presumed degradation product of mCherry-CL1, present to a variable extent when degradation is impaired. β-actin served as a loading control. E. Confocal microscopy of HeLa mCherry-CL1 cells treated with or without 100 nM bortezomib for 6 h. Scale bar, 10 μm. F. KBM7 forward genetic screen with a mCherry-CL1-expressing clone. The cells were mutagenised with the Z-loxP-GFP gene-trap retrovirus, enriched for mCherryHIGH cells by FACS, and insertion sites identified by MiSeq Illumina sequencing. G. Bubble plot of enriched genes in the mCherryHIGH population compared to unsorted mutagenised control KBM7 cells. Bubble size is proportional to the number of independent inactivating gene-trap integrations identified (shown in brackets). Genes that were significantly enriched (> −log(p)5) are shown (SPEN is found to be frequently enriched for gene-trap insertions in these types of screens and was not taken forward for further validation). H. Location of the enriched gene-trap insertions in AUP1, UBE2G2, TRC8 and MARCH6 genes (red, sense insertion; blue, antisense insertion). The predominance of insertions in the correct orientation at the start of the gene indicates enrichment for gene-trapping mutations. I. Schematic of AUP1, UBE2G2, TRC8 and MARCH6 in the ER membrane. The position of the E3 ligase RING domain is shown. SSC = side scatter. Download figure Download PowerPoint The genetic screen was performed by insertional mutagenesis of an mCherry-CL1 KBM7 clone with a gene-trapping retrovirus, and selective enrichment of high mCherry fluorescence (mCherryHIGH) by two rounds of fluorescence-activated cell sorting (FACS) (Fig 1F). Gene-trapping insertions were identified by Illumina sequencing in the mCherryHIGH population and compared to a control library of gene-trap mutagenised cells that had not been phenotypically selected (Fig 1G and Dataset EV1). Five genes were highly enriched for trapping insertions (Fig 1G and H), including two ubiquitin E3 ligases, TRC8 and MARCH6; the ubiquitin E2 conjugating enzyme, UBE2G2; and the ubiquitin-binding protein, AUP1. The fifth gene, SPEN, is frequently enriched by this type of screening approach and is not specific to the mCherry-CL1 reporter. UBE2G2 and AUP1 (Fig 1I) are orthologues of yeast Ubc7p and Cue1p and form a complex that promotes ubiquitination at the ER membrane through an E2-binding domain on AUP1 (G2BR on AUP1, U7BR on Cue1p) 8, 34, 35. Interestingly, Ubc7p and Cue1p were shown to be required for the degradation of Ura3p-CL1 in yeast 23. However, in contrast to the studies in yeast where the MARCH6 homologue, Doa10p, is sufficient for degradation of CL1 fusion proteins, the human genetic screen identified both TRC8 and MARCH6, predicting a non-redundant role for these two ER-resident E3 ligases (Fig 1I) in the degradation of mCherry-CL1 in human cells. Combined depletion of both MARCH6 and TRC8 stabilises mCherry-CL1 to levels observed with proteasome inhibition We validated a role for MARCH6, TRC8, UBE2G2 and AUP1 in the degradation of mCherry-CL1 using CRISPR/Cas9 depletion. HeLa cells were transiently transfected with Cas9 and sgRNA to all four genes and mCherry-CL1 fluorescence measured by flow cytometry on day 7. Depletion of each gene increased levels of mCherry-CL1 fluorescence but never reached the control levels observed with proteasome inhibition (Fig 2A). These findings were specific to ER ligases identified in the screen, as depletion of another ER ligase, gp78, had no effect on mCherry-CL1 levels (Appendix Fig S1A and B). Figure 2. Depletion of both MARCH6 and TRC8 is required for the stabilisation of mCherry-CL1 A. HeLa mCherry-CL1 cells were transiently transfected with Cas9 and sgRNA targeting AUP, UBE2G2, TRC8 or MARCH6, and mCherry fluorescence measured by flow cytometry after 7 days (blue). 20 nM bortezomib 16 h was used as a control for mCherry-CL1 stabilisation (brown line). B. Representative mCherry-CL1 levels (green) in isolated KO clones for AUP1, UBE2G2, TRC8 and MARCH6 from the sgRNA targeted cells (A). C. Mixed populations (shaded blue) and clonal population (shaded green) of combined TRC8/MARCH6 KO cells. 20 nM bortezomib 16 h was used as a control for mCherry-CL1 stabilisation (brown line). D. Representative immunoblot for mCherry levels in the AUP1, UBE2G2, MARCH6 and TRC8 in the HeLa mCherry-CL1 KO clones. Antibodies for AUP1, UBE2G2 and TRC8 confirmed loss of protein in the relevant clone. SQLE levels are shown in support of MARCH6 deficiency. β-actin served as a loading control. E, F. [35S]methionine/cysteine-radiolabelling of mCherry-CL1 cells in wild-type (WT), MARCH6, TRC8 or combined MARCH6/TRC8 null cells. Cells were pulse-radiolabelled for 10 min, and mCherry immunoprecipitated from detergent lysates at the times indicated. mCherry-CL1 levels were measured by autoradiography (E) and quantification (using ImageJ and GraphPad Prism) of mCherry-CL1 degradation is shown (F). Mean ± SEM, n = 3. Download figure Download PowerPoint Knockout (KO) clones isolated from the CRISPR targeted populations confirmed that specific loss of MARCH6, TRC8 E3, UBE2G2 or AUP1 increased mCherry-CL1 fluorescence and protein levels (Fig 2B–D). Due to the lack of a suitable MARCH6 antibody, a functional clonal depletion of MARCH6 was validated through the stabilisation of squalene monooxygenase (SQLE), a previously reported MARCH6 substrate 16, 36, and the presence of indel formation within the targeted MARCH6 locus (Fig 2D, Appendix Fig S2). Reconstitution of the UBE2G2, TRC8 and MARCH6 null clones with the respective wild-type cDNA restored mCherry-CL1 degradation, while catalytically inactive forms of UBE2G2 (UBE2G2 C89A), TRC8 (TRC8 ΔRING) or MARCH6 (MARCH6 C9A) had no effect on mCherry-CL1 levels (Appendix Fig S3A–F), further confirming the specificity of the gene KOs. Moreover, overexpression of the catalytically inactive enzymes (UBE2G2 C89A, TRC8 ΔRING or MARCH6 C9A) in mCherry-CL1 HeLa cells stabilised the fluorescent protein (Appendix Fig S3B, D and F), demonstrating a dominant negative effect of these mutants. Reconstitution of the AUP1 null clones with wild-type AUP1 did not restore mCherry-CL1 degradation, principally due to the toxicity of ectopic AUP1. However, it was likely that AUP1 loss resulted in destabilisation of UBE2G2 (Fig 2D, Appendix Fig S3G), similar to the S. cerevisiae orthologues, Cue1p and Ubc7p 37. The incomplete rescue of mCherry-CL1 following TRC8 or MARCH6 depletion (Fig 2A) was consistent with a requirement for both ligases. To explore this further, we measured the rate of mCherry-CL1 degradation by [35S]methionine/cysteine-radiolabelling in MARCH6 or TRC8 null cells (Fig 2E and F). mCherry-CL1 was rapidly degraded in wild-type HeLa cells with a half-life of 21 min (± 5 min) (Fig 2E and F). mCherry-CL1 degradation was also decreased in both the MARCH6 null and TRC8 null cells, but neither completely stabilised the protein (MARCH6 KO half-life 42 min ± 18, TRC8 KO half-life 62 min ± 20; Fig 2E and F). Only when both ligases were depleted did we observe an increase in mCherry-CL1 to the equivalent level seen following proteasome inhibition (Fig 2C and D) and stabilisation of the protein (Fig 2E and F). Both the MARCH6 and TRC8 catalytically inactive mutants failed to restore mCherry-CL1 degradation in the respective null clones (Appendix Fig S3), demonstrating a requirement for their ligase activity. To confirm that the ligases altered CL1 ubiquitination, we measured the accumulation of polyubiquitinated mCherry-CL1 following proteasome inhibition (MG132, 50 μM for 2 h) in wild-type or combined MARCH6/TRC8 null cells (Appendix Fig S4A). There was a marked reduction in polyubiquitinated mCherry-CL1 in the MARCH6-/TRC8-deficient cells compared to the HeLa controls (Appendix Fig S4A). The low level of residual ubiquitination in the combined null cells may reflect MARCH6- or TRC8-independent ubiquitination, but this did not seem to significantly contribute to mCherry-CL1 degradation (Fig 2F). To determine which polyubiquitin linkages were involved in CL1 degradation, we used wild-type ubiquitin and ubiquitin lysine (K) mutants, encoding a GFP fusion that is co-translationally cleaved, similar to endogenous ubiquitin processing 38, 39. Therefore, GFP levels provide a quantitative marker of ubiquitin expression and allow gating of a GFPHIGH population by flow cytometry, so that only those cells in which mutant ubiquitin outcompetes endogenous ubiquitin are analysed (Appendix Fig S4B and C). mCherry-CL1 levels increased in cells overexpressing a ubiquitin mutant incapable of forming K48 linkages (Ub-K48R), but showed no change in cells expressing Ub-K63R or Ub-K11R mutants (Appendix Fig S4C). Moreover, mCherry-CL1 levels were rescued using a K48-only ubiquitin construct compared to a Ub-K0 mutant (all lysines mutated to arginines; Appendix Fig S4B). Similar findings were observed when these ubiquitin mutants were expressed in MARCH6 or TRC8 null clones (Appendix Fig S4B and C), consistent with both ligases forming K48-ubiquitin linkages. Degradation of mCherry-CL1 by MARCH6 or TRC8 is dependent on the hydrophobicity and membrane association of its amphipathic helix Our findings suggested that ubiquitination and degradation of mCherry-CL1 were initiated at the ER membrane, but the CL1 degron is typically used as a soluble cytosolic proteasome reporter in human cells. Confocal microscopy confirmed that mCherry-CL1 was diffusely distributed throughout the cell following proteasome inhibition (Figs 1E and 3A), consistent with prior reports 22. However, some ER co-localisation was observed (Fig 3A) and we therefore probed subcellular fractionations to determine whether mCherry-CL1 associated with membranous compartments following proteasome inhibition. mCherry-CL1 was predominantly detected in the soluble (cytosolic) fraction at steady state but following proteasome inhibition, the degron was stabilised and equally distributed in the cytosol and membrane fractions (Fig 3B). Similar findings were observed in HeLa mCherry-CL1 AUP1, UBE2G2, TRC8 and MARCH6 null cells (Fig 3C), with all four KO lines showing an increase in the membrane fraction of mCherry-CL1 compared to the wild-type HeLa mCherry-CL1 lysates (Fig 3C). Figure 3. MARCH6 and TRC8 degrade membrane-associated mCherry-CL1 A. Confocal microscopy of mCherry-CL1 HeLa cells treated with 20 nM bortezomib for 16 h. A KDEL antibody was used as an ER marker. Scale bar, 10 μm. B, C. Membrane fractionation studies for mCherry-CL1 levels in HeLa mCherry-CL1 cells with or without proteasome inhibition (20 nM bortezomib 16 h) (B), or in AUP1, UBE2G2, MARCH6 or TRC8 KO HeLa mCherry-CL1 clones (C). Briefly, cells were lysed by ball-bearing homogenisation in a sucrose buffer, and the supernatant was ultracentrifuged at 50,000 rpm for 1 h to obtain the cytosol (C) and membrane (M) fractions. Tubulin and calnexin were used as control for the cytosolic and membrane fractions, respectively. D–G. Ectopic expression of TRC8 (D, E) or MARCH6 (F, G) in combined MARCH6/TRC8 null cells. Catalytically inactive mutants (MARCH6 C9A-HA and TRC8ΔRING-HA) were also overexpressed in the MARCH6/TRC8 null cells. mCherry-CL1 levels were measured by flow cytometry and gated for HA-positive cells (black line) (D, F). Basal mCherry-CL1 levels in the parent reporter cells (red) and combined MARCH6/TRC8 null cells (green) are shown. HA-tagged overexpressed ligases were also visualised by immunoblot (E, G). Download figure Download PowerPoint Proteins with exposed hydrophobic domains are often bound by chaperones to assist folding and promote degradation. Chaperones are also required to deliver transmembrane proteins to the ER and facilitate their insertion. The chaperone Bag6 (also known as BAT3 or Scythe), which forms part of the guided entry of tail-anchored proteins (GET)/transmembrane recognition complex (TRC), binds CL1 and is implicated in the recognition and subsequent ubiquitination of this degron 40. Neither Bag6 nor other chaperones were identified in the genetic screen, but these screens are unlikely to reach saturation. We therefore depleted cells of Bag6 and visualised mCherry levels by flow cytometry (Appendix Fig S5A and B). Bag6 depletion destabilised the GET/TRC pathway, as evidenced by decreased Ubl4A levels 41 (Appendix Fig S5C), but did not affect mCherry-CL1 levels (Appendix Fig S5A and B). These data are consistent with proteasome-mediated degradation of mCherry-CL1, being dependent on its ER membrane association but independent of Bag6. The partial stabilisation of mCherry-CL1 at the membrane by MARCH6 or TRC8 depletion suggested they have overlapping functions but act independently. Differences in mCherry-CL1 stabilisation were unlikely to be due to the type of ubiquitin chain formed, as both ligases showed a requirement for forming K48-ubiquitin linkages (Appendix Fig S4B and C) However, consistent with their overlapping functions, ectopic expression of either MARCH6 and TRC8 decreased the fluorescent reporter levels in the combined MARCH6-/TRC8-deficient cells (Fig 3D–G). Indeed, overexpression of TRC8 completely restored mCherry-CL1 to basal levels (Fig 3D and E) (it was possible that MARCH6 only partially restored mCherry-CL1 degradation due to its rapid autoubiquitination and degradation 42). We next explored whether the hydrophobicity of the CL1 amphipathic helix could explain the overlapping functions or specificity of the ligases. Several mutations were introduced by substituting alanine for hydrophobic residues within the CL1 amphipathic sequence (CL1 2A, CL1 4A and CL1 6A) to generate mCherry-CL1 constructs with varying degrees of hydrophobicity (Fig 4A). Stable expression of these fluorescent CL1 reporters in HeLa cells showed that their protein levels increased as hydrophobicity decreased, with mutations of all the bulky hydrophobic residues (CL1 6A) resulting in complete stabilisation of the reporter (Fig 4B, bottom panel). Moreover, as the hydrophobicity of the CL1 reporters decreased, their membrane association also decreased (Fig 4C). Indeed, once the CL1 protein was fully soluble and not hydrophobic (CL1 6A), it no longer associated with membranes and was not degraded (Fig 4C, bottom panel). Thus, CL1 hydrophobicity correlated with membrane association and proteasome-mediated degradation. Figure 4. CL1 hydrophobicity determines the specificity for recognition by MARCH6 or TRC8 A, B. HeLa cells stably expressing wild-type mCherry-CL1 or mCherry-CL1 mutants with alanine (red) mutations of the bulky hydrophobic residues (blue) (A) were transiently transfected with Cas9 and sgRNA targeting MARCH6, TRC8 or both (B). Hydrophobicity (arbitrary units) generated using "helical wheel projection" created by Don Armstrong and Raphael Zidovetzki (Version Id: wheel.pl, v 1.4 2009-10-20 21:23:36 don Exp.). Helical wheel generated using HeliQuest 75 (A). mCherry-CL1 wild-type and mutant levels (red) were measured following sgRNA transfection for the ligases (blue) or after treatment with bortezomib (Btz) (brown line) (B). C. Membrane fractionation studies for mCherry-CL1 wild-type or CL1 mutants (CL1 4A, 2A and 6A) as previously described. D. HeLa cells stably expressing the mCherry-CL1 K3A mutant were transiently transfected with Cas9 and sgRNA targeting MARCH6, TRC8 or both as described. Download figure Download PowerPoint To determine whether the CL1 mutants with decreased hydrophobicity were still degraded by MARCH6 or TRC8, we measured their mCherry levels in mixed CRISPR KO populations of each ligase, or following combined MARCH6/TRC8 depletion (Fig 4B). The unmodified mCherry-CL1 increased following TRC8 or MARCH depletion but only stabilised to the level of proteasome inhibition following combined TRC8/MARCH6 loss (Fig 4B, top panel), consistent with our prior results. However, mutations of the bulky hydrophobic residues altered the ligase specificity for CL1 degron, such that both the CL1 2A and 4A mutations were degraded solely in a TRC8-dependent manner (Fig 4B, middle panels). Moreover, depletion of TRC8 was sufficient to stabilise mCherry-CL1 2A and 4A to the same level as proteasome inhibition, with no additional change in mCherry levels observed following combined MARCH6/TRC8 loss (Fig 4B, middle panels). TRC8 or MARCH6 depletion had no effect on the levels of mCherry-CL1 6A (Fig 4B, bottom panel), but this was expected as the CL1 reporter no longer associated with membranes (Fig 4C, bottom panel). Lastly, we mutated the single lysine residue within the CL1 amphipathic helix (CL1 K3A) to determine whether this was required for ubiquitination or whether the charged residue was important for degradation (Fig 4D). The mCherry-CL1 K3A reporter was still rapidly degraded in HeLa cells, indicating that the lysine was not required for degradation (Fig 4D). Moreover, depletion of both MARCH6 and TRC8 was required for full stabilisation of mCherry-CL1 K3A, similar to the unmodified CL1 helix (Fig 4B, top panel and 4D). Thus, while both MARCH6 and TRC8 recognise the hydrophobic membrane-associated region of the CL1 degron, the degree of hydrophobicity is important, with MARCH6 requiring the longer hydrophobic region of the CL1 sequence, compared to TRC8, which can degrade the less hydrophobic CL1 mutant reporters. AUP1, UBE2G2 and TRC8 facilitate the degradation of the mCherry-CL1 through a shared pathway The incomplete rescue of mCherry-CL1 degradation in the AUP1 or UBE2G2 KO clones implied that AUP1 and UBE2G2 functioned with only one of the two ligases identified in the genetic screen. To determine the correct E2/E3 pairing, we depleted cells of AUP1 or UBE2G2 in combination with TRC8 or MARCH6 and determined which double gene KO provided a complete recovery of mCherry-CL1 fluorescence (Fig 5A and B). No combination of sgRNA targeting TRC8 with AUP1 or UBE2G2 led to a complete mCherry-CL1 recovery, as fluorescence was never increased further than observed for the single-gene knockouts (Fig 5A). However, depletion of MARCH6 in combination with AUP1 or UBE2G2 rescued mCherry-CL1 to levels seen with proteasome inhibition or combined MARCH6/TRC8 depletion (Fig 5B). These genetic data suggest that UBE2G2 and AUP1 functioned within the same pathway as TRC8. Figure 5. AUP, UBE2G2 and TRC8 facilitate the degradation of mCherry-CL1 through a shared pathway HeLa mCherry-CL1 cells (shaded red) were transiently transfected with Cas9 and combinations sgRNA targeting AUP1, UBE2G2 or TRC8, generating mixed KO populations of single genes or combinations (AUP1/UBE2G2, TRC8/AUP1 or TRC8/UBE2G2, shown in shaded blue). mCherry levels were measured by flow
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