Endoplasmic reticulum stress differentially inhibits endoplasmic reticulum and inner nuclear membrane protein quality control degradation pathways

Bryce W Buchanan,Adrian B Mehrtash,Courtney L Broshar,Avery M Runnebohm,Brian J Snow,Laura N Scanameo,Mark Hochstrasser,Eric M Rubenstein

doi:10.1074/jbc.ra119.010295

Bryce W Buchanan, Adrian B Mehrtash + Show 6 more

Open Access

https://doi.org/10.1074/jbc.ra119.010295

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Abstract

Endoplasmic reticulum (ER) stress occurs when the abundance of unfolded proteins in the ER exceeds the capacity of the folding machinery. Despite the expanding cadre of characterized cellular adaptations to ER stress, knowledge of the effects of ER stress on cellular physiology remains incomplete. We investigated the impact of ER stress on ER and inner nuclear membrane protein quality control mechanisms in Saccharomyces cerevisiae. We analyzed the turnover of substrates of four ubiquitin ligases (Doa10, Rkr1/Ltn1, Hrd1, and the Asi complex) and the metalloprotease Ste24 in induced models of ER stress. ER stress did not substantially impact Doa10 or Rkr1 substrates. However, Hrd1-mediated destruction of a protein that aberrantly engages the translocon (Deg1-Sec62) and substrates with luminal degradation signals was markedly impaired by ER stress; by contrast, Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by ER stress. ER stress impaired the degradation of one of two Asi substrates analyzed and caused a translocon-clogging Ste24 substrate to accumulate in a form consistent with persistent translocon occupation. Degradation of Deg1-Sec62 in the absence of stress and stabilization during ER stress were independent of four ER stress-sensing pathways. Our results indicate ER stress differentially impacts degradation of protein quality control substrates, including those mediated by the same ubiquitin ligase. These observations suggest the existence of additional regulatory mechanisms dictating substrate selection during ER stress.

Highlights

Hrd1-dependent degradation of proteins with intramembrane degrons was largely unperturbed by endoplasmic reticulum (ER) stress
Deg1-Sec62 becomes progressively modified by glycosylation, causing the protein to migrate as multiple species by SDS-PAGE [25] (Fig. 1B)
Our results reveal that ER stress differentially impacts ER and inner nuclear membrane (INM) protein quality control proteolytic pathways

Summary

RESULTS

N-terminal fusion of the Deg degron from MATa2 to Sec converts the protein to a Hrd ERAD-T substrate [25]. In contrast to ERAD-T and -L substrates, degradation of 6mycHmg (Fig. 2D) and Pdr5*-HA (Fig. 2E) was largely insensitive to DTT and tunicamycin; degradation of Deg1-Sec expressed in the same cells was markedly impaired during ER stress. These substrates were substantially stabilized by loss of Ubc, the primary ubiquitin-conjugating enzyme that functions with Hrd1 [4]. Deg1-Sec expression caused a ~2fold induction of the UPR in ubc7D cells, consistent with exacerbation of ER stress by persistent translocon engagement (Fig. 7). Induction of oxidative stressresponsive GFP-tagged Rtc3 [67] in a parallel culture confirmed hydrogen peroxide activity (Fig. 11D)

DISCUSSION

EXPERIMENTAL PROCEDURES

FIGURES AND FIGURE LEGENDS