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Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation.

ABSTRACT: Endoplasmic reticulum-associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome. The accumulation of misfolded proteins can be catastrophic at both the cellular and organismal level. Although the players involved and mechanistic details of ERAD are being characterized, much remains to be learned. Because of the complexity of the pathway, experimental studies generally require labor-intensive biochemical techniques. Here, we report the development of a system to analyze ERAD based on mutants of split or intact Venus fluorescent protein for which fluorescence depends on enzymatic deglycosylation. We have generated variants that only become fluorescent when they are first glycosylated in the ER and subsequently deglycosylated after retrotranslocation into the cytosol. The E3 ubiquitin ligase HMG-coA reductase degradation 1 homolog (Hrd1) and, consistent with the demonstrated glycosylation/deglycosylation requirement, the cytosolic deglycosylating enzyme peptide:N'glycanase are both required for fluorescence. Furthermore, although these deglycosylation-dependent fluorescent proteins are themselves ERAD substrates, they can also be fused to additional ERAD substrates to interrogate substrate-specific pathways. To validate the system we performed a genomewide siRNA screen that successfully identified known ERAD factors such as Hrd1; homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP); sel-1 suppressor of lin-12-like (SEL1L); and p97. These tools should greatly facilitate the identification of ERAD components and investigation of the mechanisms involved in this critical pathway.

SUBMITTER: Grotzke JE

PROVIDER: S-EPMC3587246 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation.

Grotzke Jeff E JE Lu Qiao Q Cresswell Peter P

Proceedings of the National Academy of Sciences of the United States of America 20130211 9

Endoplasmic reticulum-associated degradation (ERAD) is a constitutive process that identifies misfolded proteins in the ER and shuttles them to the cytosol, where they can be degraded by the proteasome. The accumulation of misfolded proteins can be catastrophic at both the cellular and organismal level. Although the players involved and mechanistic details of ERAD are being characterized, much remains to be learned. Because of the complexity of the pathway, experimental studies generally require ...[more]

PMID: 23401531

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Project description:Abnormalities in posttranslational protein modifications (PTMs) that regulate protein targeting, trafficking, synthesis, and function have been implicated in the pathophysiology of schizophrenia. The endoplasmic reticulum (ER) contains specialized machinery that facilitate protein synthesis, ER entry and exit, quality control, and post-translational processing, steps required for protein maturation. Dysregulation of these systems could represent potential mechanisms for abnormalities of neurotransmitter associated proteins in schizophrenia. We hypothesized that expression of ER processing pathways is dysregulated in schizophrenia. We characterized protein and complex expression of essential components from protein folding, ER quality control (ERQC), and ER associated degradation (ERAD) processes in the dorsolateral prefrontal cortex of 12 matched pairs of elderly schizophrenia and comparison subjects. We found increased expression of proteins associated with recognizing and modifying misfolded proteins, including UDP-glucose/glycoprotein glucosyltransferase 2 (UGGT2), ER degradation enhancing alpha-mannosidase like protein 2 (EDEM2), and synoviolin (SYVN1)/HRD1. As SYVN1/HRD1 is a component of the ubiquitin ligase HRD1-SEL1L complex that facilitates ERAD, we immunoprecipitated SEL1L and measured expression of other proteins in this complex. In schizophrenia, SYVN1/HRD1 and OS-9, ERAD promoters, have increased association with SEL1L, while XTP3-B, which can prevent ERAD of substrates, has decreased association. Abnormal expression of proteins associated with ERQC and ERAD suggests dysregulation in ER localized protein processing pathways in schizophrenia. Interestingly, the deficits we found are not in the protein processing machinery itself, but in proteins that recognize and target incompletely or misfolded proteins. These changes may reflect potential mechanisms of abnormal neurotransmitter associated protein expression previously observed in schizophrenia.

Dataset Information

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation.

Publications

Deglycosylation-dependent fluorescent proteins provide unique tools for the study of ER-associated degradation.

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