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.

Sec61 blockade by mycolactone inhibits antigen cross-presentation independently of endosome-to-cytosol export

“…Notably, accessory molecules that contribute to ERAD of the substrates used in this study [HERP, AUP1, and FAF2/UbxD8 (22,25)] were up-regulated. These proteins likely belong to the small subset of Sec61 clients resisting mycolactone inhibitory activity, and their accumulation in treated DCs may reflect a stress response to ER translocation defects (13,16).…”

“…We next investigated whether mycolactonemediated Sec61 inhibition affected the export of proteins from the ER into the cytosol during ERAD. To achieve this goal, we used the previously described deglycosylation-dependent (dd) ERAD substrate constructs ddVenus and ddSplit Venus (22) as fluorescent reporters of ERAD activity ( Fig. 2A).…”

Approaches to imaging unfolded secretory protein stress in living cells

“…Grotzke et al identified mutant variants of the FP Venus that can only become fluorescent when first glycosylated in the ER and then deglycosylated in the cytoplasm following ERAD retrotranslocation [93]. The reporter, termed ddVenus (for deglycosylation dependent) [93], exploits the conversion of glycosylated asparagines to aspartates by PNGaseF in the cytoplasm.…”

“…Grotzke et al identified mutant variants of the FP Venus that can only become fluorescent when first glycosylated in the ER and then deglycosylated in the cytoplasm following ERAD retrotranslocation [93]. The reporter, termed ddVenus (for deglycosylation dependent) [93], exploits the conversion of glycosylated asparagines to aspartates by PNGaseF in the cytoplasm. By expressing half of ddVenus in the ER and the other half in the cytoplasm, a highly specific ERAD-dependent fluorescent signal can be achieved and quantitated by both fluorescence microscopy and flow cytometry [93].…”

“…The reporter, termed ddVenus (for deglycosylation dependent) [93], exploits the conversion of glycosylated asparagines to aspartates by PNGaseF in the cytoplasm. By expressing half of ddVenus in the ER and the other half in the cytoplasm, a highly specific ERAD-dependent fluorescent signal can be achieved and quantitated by both fluorescence microscopy and flow cytometry [93]. …”