Acetylation of N-terminus and two internal amino acids is dispensable for degradation of a protein that aberrantly engages the endoplasmic reticulum translocon

Abstract

Conserved homologues of the Hrd1 ubiquitin ligase target for degradation proteins that persistently or aberrantly engage the endoplasmic reticulum translocon, including mammalian apolipoprotein B (apoB; the major protein component of low-density lipoproteins) and the artificial yeast protein Deg1-Sec62. A complete understanding of the molecular mechanism by which translocon-associated proteins are recognized and degraded may inform the development of therapeutic strategies for cholesterol-related pathologies. Both apoB and Deg1-Sec62 are extensively post-translationally modified. Mass spectrometry of a variant of Deg1-Sec62 revealed that the protein is acetylated at the N-terminal methionine and two internal lysine residues. N-terminal and internal acetylation regulates the degradation of a variety of unstable proteins. However, preventing N-terminal and internal acetylation had no detectable consequence for Hrd1-mediated proteolysis of Deg1-Sec62. Our data highlight the importance of empirically validating the role of post-translational modifications and sequence motifs on protein degradation, even when such elements have previously been demonstrated sufficient to destine other proteins for destruction.

Overlapping function of Hrd1 and Ste24 in translocon quality control provides robust channel surveillance

“…Roles for Hrd1 and Ste24 in TQC have been inferred by their ability to target unique model substrates engineered to aberrantly engage the translocon. Hrd1 substrates are incompletely stabilized in hrd1D yeast, suggesting parallel degradative mechanisms (19). To query their specificity in TQC, we asked if Hrd1 and Ste24 can target characterized substrates of the other enzyme.…”

“…Cycloheximide chase experiments (48), cell lysis, western blotting (49,50), and endoglycosidase H (Endo H; New England Biolabs) treatment (19) were performed as described. The following antibody dilutions were used: mouse anti-HA.11 (Clone 16B12; BioLegend) at 1:1,000 to detect Clogger; mouse anti-c-myc (Clone 9E10; BioLegend) at 1:1,000 to detect sec61-13myc; and mouse antiphosphoglycerate kinase 1 (Pgk1; clone 22C5D8; Thermo Fisher Scientific) at 1:20,000.…”

The Relationship between ER Stress and Protein Quality Control at the Translocon

“…These mutations did not affect Hrd1-mediated degradation of Deg1-Sec62 suggesting that glycosylation was not required for degradation [10]. Degradation of variants of Deg1*-Sec62 with mutated acetylation sites showed that acetylation was also not required for Hrd1-dependent degradation of Deg1*-Sec62 [25]. There may be uncharacterized PTMs that contribute to Deg1-Sec62 degradation, but there is no evidence to suggest so.…”

“…ER stress does not inhibit Hrd1 function directly, since degradation of some Hrd1-targeted proteins (such as ERAD-M substrates) is unaffected by ER stress. While ER stress impairs modification of translocon-clogging proteins [45] this is likely not the reason for impaired degradation, since it has been shown modifications are not required for degradation [10,25]. Inhibition of ERAD-L by ER stress is likely to occur by saturation of the multifunctional ER chaperone Kar2 [7]; Kar2 overexpression partially rescues impaired ERAD-L during conditions associated with elevated ER stress [38].…”

“…Glycosylation is also required for degradation of a subset of Hrd1 substrates [10]. It has been shown however, that neither glycosylation nor acetylation are needed for Deg1-Sec62 degradation [10,25]. Nglycosylation sites were mutated (N90D and N153D) in cells expressing Deg1-Sec62.…”