Protein quality control at the inner nuclear membrane

Abstract

The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression1. The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitin-conjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer5, we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

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Figure 1: The Asi complex is a Ubc6/Ubc7-dependent E3 ubiquitin ligase of the INM.
Figure 2: Functional overlap between Asi and ERAD E3 ubiquitin ligases.
Figure 3: Systematic identification of substrates for Asi and ERAD E3 ubiquitin ligases.

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Acknowledgements

We thank M. Lemberg, E. Schiebel and B. Bukau for support and discussions, A. Kaufmann, C.-T. Ho, A. Bartosik and B. Besenbeck for help with tFT library construction, K. Ryman for the qRT–PCR analysis of gene expression, M. Hochstrasser for strains, the GeneCore and the media kitchen facilities of the European Molecular Biology Laboratory (EMBL) and Donnelly Centre for support with infrastructure and media. This work was supported by the Sonderforschungsbereich 1036 (SFB1036, TP10) from the Deutsche Forschungsgemeinschaft (DFG) (M.K.), the Swedish Research Council grant VR2011-5925 (P.O.L.), INSERM and grants from ANR (ANR-12-JSV8-0003-001) and Biosit (G.R.), fellowships from the European Molecular Biology Organization (EMBO ALTF 1124-2010 and EMBO ASTF 546-2012) (A.K.) and fellowships from the Ministère de la Recherche et de l’Enseignement Supérieur and La Ligue Contre le Cancer (E.B.). M.K. received funds from the CellNetworks Cluster of Excellence (DFG) for support with tFT library construction. W.H. acknowledges funding from the EC Network of Excellence Systems Microscopy. C.B. was supported by funds from the Canadian Institute for Advanced Research (GNE-BOON-141871), National Institutes of Health (R01HG005853-01), Canadian Institute for Health Research (MOP-102629) and the National Science and Engineering Research Council (RGPIN 204899-6).

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Contributions

G.R. designed the BiFC, microscale thermophoresis and ubiquitin pulldown experiments that were performed by E.B., G.L.D. and A.B. M.P., D.J.O. and A.G. contributed the biochemical analysis of Asi-dependent ubiquitylation. M.K. and A.K. designed and coordinated the tFT project. A.K. and M.M. designed and constructed the tFT library and performed the screens with help from D.K. and C.B. B.F. and J.D.B. developed the screen analysis methods, with input from A.K., M.K., W.H. and C.B. M.K., A.K., G.R. and P.O.L. prepared the figures and wrote the paper with input from all authors.

Corresponding authors

Correspondence to Gwenaël Rabut or Per O. Ljungdahl or Michael Knop.

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Extended data figures and tables

Extended Data Figure 1 Identification of Ubc6 and Ubc7 ubiquitin-conjugating enzymes as functional interacting partners of Asi1 and Asi3.

a, Quantification of BiFC signals in cells expressing VC–Ubc6 and all tested E3 ubiquitin ligases. BiFC signals were measured in the cytoplasm and nucleus of individual cells (n as shown). Whiskers extend from the tenth to the ninetieth percentiles. The same representation is used in c and d. b, Immunoblot showing expression levels of VC-tagged E2 ubiquitin-conjugating enzymes. Ubc11–VC could not be detected in the growth condition of the BiFC assay. c, Quantification of BiFC signals in cells co-expressing VC-tagged E2 ubiquitin-conjugating enzymes and Asi1–VN or Asi3–VN (n as shown). d, Detection of a significant BiFC signal between Asi1–VN and Ubc4–VC in cells lacking UBC6 (n as shown). e, Coomassie-stained gels of recombinant proteins used in microscale thermophoresis experiments. f, mRNA levels of AGP1 and GNP1 measured with qRT–PCR in the indicated strains (mean ± s.d., n = 3 clones). The signal was normalized to wild type (dashed line). g, Ubiquitylation of Stp1–HA or Stp1-RI17–33–HA (Stp1 variant in which amino acid residues 2–64 were replaced with Stp1 residues 17–33 flanked by minimal linker sequences) (left) and Stp2–HA or Stp2Δ2–13–HA (Stp2 variant lacking amino acid residues 2–13) (right) in strains expressing 6×His–ubiquitin. Stp1-RI17–33 and Stp2Δ2–13 variants exhibit compromised cytoplasmic retention and enhanced Asi-dependent degradation, whereas full-length Stp1 is degraded primarily in the cytoplasm in a SCFGrr1-dependent manner11. Total cell extracts (T), flow-through (F) and ubiquitin conjugates (E) eluted after immobilized-metal affinity chromatography were separated by SDS–PAGE followed by immunoblotting with antibodies against the HA-tag, Pgk1 and the His-tag. Representative immunoblots from three technical replicates. *P < 10−4 (a, c and d; one-way ANOVA with Bonferroni correction for multiple testing), and *P < 0.05, **P < 0.1 (f; two-tailed t-test).

Extended Data Figure 2 Lack of genetic interaction between ASI1 and HRD1 or DOA10 at 37 °C.

Tenfold serial dilutions of strains grown on synthetic complete medium for 2 days at 30 or 37 °C.

Extended Data Figure 3 tFT screens for substrates of Asi and ERAD E3 ubiquitin ligases.

a, Tagging approach used to construct the tFT library in a strain carrying the I-SceI meganuclease under an inducible promoter. First, a module for seamless C-terminal protein tagging with the mCherry-sfGFP timer is integrated into a genomic locus of interest using conventional PCR targeting. Subsequent I-SceI expression leads to excision of the heterologous terminator and the URA3 selection marker, followed by repair of the double-strand break by homologous recombination between the mCherry and mCherryΔN sequences. A tFT fusion protein is expressed under control of endogenous promoter and terminator in the final strain. b, Workflow of screens for substrates of E3 ubiquitin ligases involved in protein degradation. Each tFT query strain is crossed to an array of mutants carrying different gene deletion alleles. The resulting strains are imaged with a fluorescence plate reader to identify proteins with altered stability in each mutant. c, Volcano plots of the screens for proteins with altered stability in the indicated mutants. Plots show z-scores for changes in protein stability on the x axis and the negative logarithm of P values adjusted for multiple testing on the y axis. The number of proteins with increased (red) or decreased (blue) stability at 1% false discovery rate is indicated. d, Fraction of proteins in the tFT library and in the three clusters in Fig. 3b mapped to the full yeast slim set of component GO terms. Note that the GO term cytoplasm contains all cellular contents except the nucleus and the plasma membrane. e, The three clusters in Fig. 3b are enriched for proteins in the indicated component GO terms. Bar plot shows −log10-transformed P values of significant enrichments.

Extended Data Figure 4 Analysis of integral membrane protein substrates of the Asi E3 ubiquitin ligase.

a, Differences in the log10mCherry/sfGFP intensity ratio between the indicated mutants and the wild type (mean ± s.d., n = 4) for tFT-tagged proteins from the Asi cluster in Fig. 3b. b, Quantification of BiFC signals in strains co-expressing VC–Ubc6 and Asi3–VN (top). BiFC signals were measured in the cytoplasm and nucleus of individual cells (n as shown). Whiskers extend from tenth to ninetieth percentiles. A substantial BiFC signal is retained in the asi2Δ mutant, despite reduced expression of Asi3 (immunoblot, bottom). c, Quantification of sfGFP signals in strains expressing tFT-tagged proteins from the Asi cluster in Fig. 3b. Fluorescence microscopy examples representative of five fields of view (top). Scale bar, 5 μm. sfGFP intensities were measured in individual cells (middle) and at the nuclear rim (bottom). For each protein, measurements were normalized to the mean of the respective wild type. Whiskers extend from minimum to maximum values. *P < 0.05 (a and c; two-tailed t-test) and *P < 10−4 (b; one-way ANOVA with Bonferroni correction for multiple testing).

Extended Data Figure 5 Cycloheximide chase experiments with substrates of the Asi E3 ubiquitin ligase.

Degradation of 3×HA-tagged proteins after blocking translation with cycloheximide. Whole-cell extracts were separated by SDS–PAGE followed by immunoblotting with antibodies against the HA tag and Pgk1 as loading control. Representative immunoblots from two technical replicates. Left, wild-type and asi1Δ immunoblots are reproduced in Fig. 3f.

Extended Data Figure 6 Influence of tagging and expression levels on localization of Vtc1 and Vtc4.

Fluorescence microscopy of strains expressing Vtc1 or Vtc4 tagged endogenously with monomeric yeast codon-optimized enhanced GFP (myeGFP) at the C terminus or tagged with sfGFP at the N terminus and expressed under control of endogenous or TEF1 promoters. Representative deconvolved images of five fields of view with 100 cells each. Arrowheads indicate nuclear rim localization. Scale bar, 5 μm.

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Khmelinskii, A., Blaszczak, E., Pantazopoulou, M. et al. Protein quality control at the inner nuclear membrane. Nature 516, 410–413 (2014). https://doi.org/10.1038/nature14096

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