Order through destruction: how ER-associated protein degradation contributes to organelle homeostasis

Abstract

The endoplasmic reticulum (ER) is a large, dynamic, and multifunctional organelle. ER protein homeostasis is essential for the coordination of its diverse functions and depends on ER-associated protein degradation (ERAD). The latter process selects target proteins in the lumen and membrane of the ER, promotes their ubiquitination, and facilitates their delivery into the cytosol for degradation by the proteasome. Originally characterized for a role in the degradation of misfolded proteins and rate-limiting enzymes of sterol biosynthesis, the many branches of ERAD now appear to control the levels of a wider range of substrates and influence more broadly the organization and functions of the ER, as well as its interactions with adjacent organelles. Here, we discuss recent mechanistic advances in our understanding of ERAD and of its consequences for the regulation of ER functions.

Keywords: ERAD; endoplasmic reticulum; protein degradation; protein quality control; ubiquitin ligase.

Figure 1. The steps and ubiquitin ligases involved in ERAD

(A) General steps involved in the ERAD of different classes of misfolded proteins (in black). Ubiquitin ligase complexes in the ER membrane promote the recognition, retrotranslocation, and ubiquitination of ERAD substrates. Recognition of misfolded glycoproteins in the ER lumen requires trimming of glycans (in red, see text for details). Ubiquitinated substrates are released from the ER membrane into the cytosol by the Cdc48/p97/VCP ATPase, trimmed, and re‐ubiquitinated before being handed to the proteasome for degradation. (B) E3 ubiquitin ligases and their cognate E2 enzymes characterized in yeast and mammalian cells. Homologous ERAD complexes in yeast and mammalian cells are shown together, separated by dotted lines. ERAD complexes exclusive to yeast and mammalian cells are show in separate boxes. Arrows indicate the subcellular environment by which substrates have been shown to access each E3.

Figure 2. Proposed mechanisms for key processing steps in ERAD

(A) Cdc48/p97/VCP ATPase (yellow) has a universal role in pulling ubiquitinated substrates from the ER membrane. Ubiquitin molecules conjugated to the substrate interact with the ATPase complex and unfold as they enter the central pore (1). Rounds of ATP hydrolysis promote the threading of the substrate through the central pore (2). As substrates emerge on the trans side of the ATPase complex, they may be re‐ubiquitinated by soluble ubiquitin ligases (3). De‐ubiquitinating enzymes (Otu1 and Yod1 in yeast and mammals, respectively) on the cis‐side remove residual ubiquitin moieties, facilitating the remaining residues of the substrate to travel through the central pore. Glycans conjugated to glycoproteins are removed by peptide N‐glycanase enzymes (Png1 and NGly1 in yeast and mammals, respectively) associated with the cis‐ or trans‐sides of the Cdc48/p97/VCP complex. (B) Proposed mechanisms for the retrotranslocation of luminal glycoproteins. In yeast (from left to right), upon recognition by Hrd3/Yos9, substrates engage with the membrane domains of Hrd1 and Der1, which form two halves of a channel. Besides being rich in hydrophilic residues, Hrd1/Der1 membrane regions promote the local thinning of the ER membrane, thereby facilitating the retrotranslocation of the substrate to the cytosolic side of the ER membrane, where it is ubiquitinated by the activity of Hrd1 RING domain (in inset). Ubiquitinated substrate is pulled through the channel and across the membrane by the Cdc48/p97/VCP as described in (A). Although less mechanistic studies are available, analogous steps are thought to occur during the retrotranslocation of luminal glycoproteins in mammals (Right to left). (C) Proposed retrotranslocation mechanism for a membrane ERAD substrate in yeast. Membrane substrates ubiquitinated by the Hrd1 or Doa10 ERAD ubiquitin ligase complexes interact with Dfm1, which facilitates their retrotranslocation with the assistance of the Cdc48/p97/VCP ATPase complex. Dfm1 can also promote retrotranslocation of ubiquitinated substrates independently of Hrd1 and Doa10. The mechanisms and the oligomeric state of Dfm1 during retrotranslocation are unclear.

Figure 3. Regulation of sterol biosynthesis by ERAD

Endoplasmic reticulum‐resident enzymes involved in sterol biosynthesis pathways of yeast (top) and mammals (bottom) are shown, along with the different ubiquitin ligases that regulate their abundance via ERAD. Sterol‐related biosynthetic products of each enzyme are shown (in blue) as are the nonsterol isoprenoids (in green). Enzymes whose regulation by ERAD has not yet been described are also shown (in red).

Figure 4. Asi‐mediated transmembrane domain recognition at the inner nuclear membrane

Selected TM domain‐containing proteins failing to assemble appropriately into oligomeric complexes in yeast can diffuse through the ER membrane (in brown) and past the nuclear pore complex (NPC) to the inner nuclear membrane (in blue). There, the Asi complex (Asi1/2/3) engages these orphan subunits through Asi2‐mediated recognition of the substrate's TM domain, leading to subsequent ubiquitination and degradation via nuclear‐localized proteasomes.

Figure 5. Regulation of organelle interfaces by ER‐resident E3s

(A) Two examples of ER‐resident ubiquitin ligase complexes regulating proteins at the ER‐mitochondria interface. Hrd1‐SEL1L impact abundance of the Sigma receptor (left) while RNF170, bridged by Erlin1/2, ubiquitinates IP3 receptors that regulate calcium ion efflux from the ER to the mitochondria through VDAC (right). (B) Regulation of endosomal positioning relative to the ER through ubiquitination of p62/SQSTM by the ER‐resident E3 RNF26 and E2 UBE2J1 (left). Ubiquitinated p62/SQSTM binds to endosomal proteins (e.g., TOLLIP, EPS15, TAX1BP1) to form a perinuclear endosomal cloud, which can be dispersed by the activity of the USP15 deubiquitinase. RNF26 is also, along with RNF5 and gp78/INSIG1, involved in ubiquitination of the innate immune signaling molecule STING when present in the ER membrane. Together with several interacting cofactors (TMEM43, TMEM33, TMED1, ENDOD1), RNF26 modulates the magnitude of interferon signaling mediated through the cGAS‐STING pathway.