The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation

Abstract

The endoplasmic reticulum (ER) serves as a warehouse for factors that augment and control the biogenesis of nascent proteins entering the secretory pathway. In turn, this compartment also harbors the machinery that responds to the presence of misfolded proteins by targeting them for proteolysis via a process known as ER-associated degradation (ERAD). During ERAD, substrates are selected, modified with ubiquitin, removed from the ER, and then degraded by the cytoplasmic 26S proteasome. While integral membrane proteins can directly access the ubiquitination machinery that resides in the cytoplasm or on the cytoplasmic face of the ER membrane, soluble ERAD substrates within the lumen must be retrotranslocated from this compartment. In either case, nearly all ERAD substrates are tagged with a polyubiquitin chain, a modification that represents a commitment step to degrade aberrant proteins. However, increasing evidence indicates that the polyubiquitin chain on ERAD substrates can be further modified, serves to recruit ERAD-requiring factors, and may regulate the ERAD machinery. Amino acid side chains other than lysine on ERAD substrates can also be modified with ubiquitin, and post-translational modifications that affect substrate ubiquitination have been observed. Here, we summarize these data and provide an overview of questions driving this field of research.

Keywords: molecular chaperone; post-translational modification; proteasome; protein quality control; ubiquitin ligases.

Figure 1. The ubiquitination pathway

The cytosolic E1 ubiquitin-activating enzyme hydrolyzes ATP to activate the ubiquitin molecule. ATP hydrolysis and the formation of a transient AMP-derivative lead to the formation of a thioester bond between the E1 and the C-terminus of ubiquitin. The E1 then transfers ubiquitin to one of the ~11 yeast or the ~35 mammalian E2 ubiquitin-conjugating enzymes. The covalently bound ubiquitin—E2 adduct then binds one of the ~80 yeast or the ~300 mammalian E3 ubiquitin ligases. The E3 enzymes may also be bound to the ERAD substrate and facilitate transfer of ubiquitin to the substrate, or a chaperone intermediate (not shown) may facilitate transfer. Some E3 ubiquitin ligases (e.g. HECT domain E3s) become covalently modified with ubiquitin during ERAD substrate modification, while other E3s (e.g. RING and U-box domain proteins) facilitate the transfer of ubiquitin from the E2 ubiquitin-conjugating enzyme to the substrate. Importantly, select E2 ubiquitin-conjugating enzymes and E3 ubiquitin ligases are cytoplasmic, while others reside at the ER membrane. Once a substrate is ubiquitinated by an E3 ubiquitin ligase, other enzymes, such as E4s, may further extend the ubiquitin chain on the ERAD substrate.

Figure 2. Function of the Hrd1 complex during ERAD in yeast

In the first step during ERAD (‘Recognition’), a misfolded substrate is recognized by a subset of factors, namely Kar2 (ER luminal Hsp70 chaperone), Yos9 (ER luminal lectin), Der1 (transmembrane core Hrd1 complex member), or directly by the E3, Hrd1. Once the substrate has been recognized, the substrate is transferred to the Hrd1 complex for polyubiquitination. Kar2 and Yos9 bind to the Hrd1 core complex member, Hrd3, and the substrate is transferred to Hrd1 (‘Ubiquitination’). Der1 is bound by Usa1, which helps link Der1 to Hrd1. Cue1 is an ER membrane protein that recruits the E2 ubiquitin-conjugating enzyme, Ubc7, to the Hrd1 complex. After the substrate is polyubiquitinated, the dislocation machinery is linked to the complex. This dislocation complex consists of the membrane protein, Ubx2, which helps recruit the AAA+ ATPase, Cdc48. Cdc48 is stabilized at the Hrd1 complex through an interaction with Hrd1 and through an interaction with the Cdc48 cofactors, Ufd1 and Npl4, with the polyubiquitin chain. Assembly is believed to be due to Ufd1 binding to the polyubiquitin chain, as yeast Npl4 lacks a zinc finger domain. Another class of cofactors that bind Cdc48 and affect retrotranslocation includes DUBS, such as Otu1 (not shown here). Once bound to the substrate, Cdc48 hydrolyzes ATP and liberates the substrate from the ER (‘Retrotranslocation’). The Cdc48 cofactor, Ufd2, then extends the polyubiquitin chain and interacts with the ubiquitinated protein shuttles, Rad23 and Dsk2 (‘Degradation’). Although not shown in this figure, ubiquitin chains may be trimmed by DUBS prior to substrate passage through Cdc48 and then extended again by Ufd2. Rad23 and Dsk2 can also interact with the 19S cap of the cytosolic 26S proteasome, which leads to substrate degradation. Another DUB associated with the 19S cap of the proteasome, called Rpn11 (not shown), removes the polyubiquitin chain attached to the ERAD substrate, so it efficiently threads into the core of the 26S proteasome for degradation.

Figure 3. Function of the Hrd1 complex during ERAD in mammalian cells

In the first step during ERAD (‘Recognition’), a substrate is recognized by a group of luminal and membrane-associated factors, namely BiP (Hsp70 chaperone), OS9 and XTP-3 (lectins), Derlin1 (transmembrane core Hrd1 complex member), and/or HRD1. While OS9 and XTP-3 recognize misfolded ER luminal glycosylated substrates, they may also recognize nonglycosylated substrates. The substrate is then transferred to the HRD1 complex and polyubiquitinated. BiP, OS9, and XTP-3 bind the HRD1 core complex member, SEL1L, and the substrate is next transferred to HRD1 (‘Ubiquitination’). DERLIN1 is bound by HERP1, which helps link Derlin1 to HRD1. The mammalian HRD1 complex can utilize DERLIN2 and DERLIN3 as well. AUP1 is a Cue domain-containing ER membrane protein that recruits the E2 ubiquitin-conjugating enzyme, UBE2G2, to the HRD1 complex. Once polyubiquitinated, the dislocation machinery is recruited to the HRD1 complex. This complex includes the membrane protein, UBXD8, which augments p97 recruitment to the HRD1 complex. Along with UBXD8, the HRD1 complex can utilize another protein, called UBXD2, to recruit p97. p97 is further stabilized at the HRD1 complex through interaction with HRD1 along with the p97 cofactors, UFD1 and NPL4, via the polyubiquitin chain. In an alternative mechanism of p97 recruitment, the cytoplasmic protein, VIMP, binds p97 (not shown) at the HRD1 complex through a VIMP interaction domain in DERLIN1. Another class of cofactors that bind p97 and act during ERAD include DUBS, such as YOD1 (not shown). Once bound to the substrate, p97 hydrolyzes ATP and removes the substrate from the ER (‘Retrotranslocation’). After retrotranslocation, the p97 cofactor and a mammalian homolog of Ufd2, UBE4A/B, may extend the polyubiquitin chain and associate with the protein shuttles, RAD23A, and a specific UBIQUILIN, UBQLN2 (‘Degradation’). Ubiquitin chains may be trimmed by DUBS prior to substrate transit through p97 and could then be extended again by UBE4A/B. RAD23A and UBIQUILIN also interact with the 19S cap of the cytosolic 26S proteasome, which facilitates substrate degradation. In addition, there is a DUB associated with the 19S cap of the proteasome, RPN11, which removes the polyubiquitin chain attached to the ERAD substrate, which aids efficient entry of the substrate into the 26S proteasome core.