Description

Approximately one third of eukaryotic proteins enter the endoplasmic reticulum (ER) en route to their subcellular or extracellular destinations (Chen et al. 2005; Choi et al. 2010). Many of these proteins use the Sec61p translocon complex to cross the ER membrane (Aviram and Schuldiner 2017). Proteins that persistently engage the translocon prevent other proteins from reaching the ER (Izawa et al. 2012; Ast et al. 2016). Thus, cells have evolved multiple quality control mechanisms to degrade proteins that aberrantly occupy this channel (Rubenstein et al. 2012; Crowder et al. 2015; Ast et al. 2016). In ER-associated degradation of translocon-associated proteins (ERAD-T), such polypeptides are targeted for destruction by homologs of the ER-resident RING (really interesting new gene) domain ubiquitin ligase Hrd1p. Deg1*-Sec62 is an engineered model translocon-associated substrate for Hrd1p in yeast (Figure 1A). Analogously, in mammalian cells, the Hrd1p homolog gp78 promotes turnover of the low-density lipoprotein (LDL) component apolipoprotein B, which stalls in the translocon if it is unable to associate with LDL lipid molecules (Fisher et al. 2011).

We recently discovered that degradation of Deg1*-Sec62 is impaired by ER stress (the accumulation of misfolded or unfolded proteins in the ER). Deg1*-Sec62 is strongly stabilized by treatment with dithiothreitol (DTT; which reduces disulfide bonds) or tunicamycin (which prevents N-linked glycosylation). By contrast, however, Deg1*-Sec62 degradation is unaffected by perturbation of ER membrane lipid composition (i.e. inositol limitation) or treatments expected to broadly perturb proteostasis (elevated temperature or oxidative stress) (Buchanan et al. 2019).

The AMP-activated protein kinase Snf1p is stimulated during ER stress (Mizuno et al. 2015). Further, loss of the Snf1p inhibitor Reg1p renders cells hypersensitive to ER stress (Ferrer-Dalmau et al. 2015). Snf1p is also regulated by nutrient abundance; it is activated by phosphorylation when glucose is limiting and inactivated by dephosphorylation when glucose is abundant (Rubenstein et al. 2008). Given ERAD-T sensitivity to ER stress and crosstalk between ER stress and nutrient stress signaling, we sought to determine if turnover of the ERAD-T substrate Deg1*-Sec62 is regulated by changes in glucose abundance.

We performed cycloheximide chase experiments to compare Deg1*-Sec62 degradation kinetics in low (0.05%), standard (2%), or high (8%) glucose concentrations (Figure 1B). Deg1*-Sec62 was rapidly degraded in all three conditions. By contrast, DTT strongly stabilized and impaired post-translational modification of Deg1*-Sec62, as previously reported (Buchanan et al. 2019). ADH2 expression is repressed by glucose (Dombek et al. 1993). To confirm differences in glucose abundance, ADH2-GFP expression was compared using flow cytometry of a parallel culture (Figure 1C). Our results indicate that changes in glucose abundance (in the range of 0.05% to 8%) do not substantially alter the rate of degradation of Deg1*-Sec62, a model translocon-associated substrate of Hrd1p.

Taken with our recently published work (Buchanan et al. 2019), our results indicate that ERAD-T is inhibited by stress caused by ER protein misfolding but not membrane stress, oxidative stress, heat shock, or glucose limitation or abundance. It remains possible that altered glucose levels exert an effect on ERAD-T in the context of ER stress or mutations in genes mediating crosstalk between ER stress and nutrient signaling. Future experiments may be performed to test these hypotheses. During ER stress, protein translocation into the ER is slowed (Kang et al. 2006). We speculate that inhibited degradation of proteins that persistently engage the translocon contributes to reduced overall rates of translocation, preventing an already stressed ER from becoming overwhelmed.