Summary- paper 6:
Dynamic quality control machinery that operates across compartmental borders mediates the degradation of mammalian nuclear membrane proteins
Pei-Ling Tsai, Christopher J.F.Cameron, Maria Fernanda Forni, Renee R.Wasko, Brigitte S.Naughton, Valerie Horsley, Mark B.Gerstein, Christian Schlieker
Cell Reports, 2022
Questions/gaps addressed:
- What are the molecular mechanisms responsible for protein folding and turnover in the inner nuclear membrane (INM)? ER is contiguous with the outer nuclear membrane (ONM), not INM, which E3 ligases surveil the INM?
Major hypotheses:
- ONM/INM diffusion barrier necessitates that a dedicated INM-resident machinery exists to perform a ERAD-like role.
Key methods:
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LBR1600 Lamin B receptor (LBR) mutant, responsible for Pelger-Huët anomaly and Greenberg skeletal dysplasiais metabolically unstable/ short lived, serves as a INM PQC model substrate.
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split-GFP system to monitor the status of mutant LBR1600 localization (last beta strand of GFP (S11) fused to its C terminus) and NLS-GFP1-10. Both expressed froma Tet-inducible retroviral system.
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GFP fluorescence stabilization as a read-out for CRISPR-Cas9-based screen (Brunello KO library- 4 sgRNAs/gene against 19,114 genes; 1,000 sgRNA controls) to identifying LBR1600 turnover machinery. Cells with sgRNAs and stabilized GFP fluorescence sorted and enriched (2 rounds), and sgRNA frequency analyzed. Proof of principle stabilization upon treatment with VCP INHIBITOR, CB-5083.
Major takeaways:
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LBR1600 turnover is stabilized in KO of E2 ubiquitin-conjugating enzymes Ube2G2 and Ube2D3, as well as the E3 Ub ligases RNF5 and HRD1/SYVN1 (confirmed by rescue and catalytic inactive mutants), and uncharacterized membrane protein TMEM33. All resulted in reduced polyubiquitination, predominandtly K48 linkage. VCP was not identified as a hit in the screen.
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RNF5-HA (CRISPR/Cas12a-mediated PCR tagging) immunoprecipitates LBR1600-GFP11. TMEM33-HA pulls down FLAG-RNF5 as well as LBR1600.
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TMEM33 works upstream of RNF5 in the same pathway for LBR1600 turnover, HRD1 acts via an independent pathway. OE of RNF5 in hrd1-KO leads to LBR1600 degradation. What about OE of HRD1 in rnf5-KO cells? Is either sufficient?
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TMEM33 is required for RNF5 protein stability- act as a chaperone for RNF5 or binding might prevent RNF5 auto-ubiquitination?
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RNF5 localizes to the ER and INM- authors propose that RNF5 is small (20kD) and may easily pass the ER-INM diffusion barrier, hence accessign INM substrates. If Hrd1 cannot pass this barrier- how then does it have an effect on LBR1600 turnover?