A structurally unique E2-binding domain activates ubiquitination by the ERAD E2, Ubc7p, through multiple mechanisms.

Cue1p is an integral component of yeast endoplasmic reticulum (ER)-associated degradation (ERAD) ubiquitin ligase (E3) complexes. It tethers the ERAD ubiquitin-conjugating enzyme (E2), Ubc7p, to the ER and prevents its degradation, and also activates Ubc7p via unknown mechanisms. We have now determined the crystal structure of the Ubc7p-binding region (U7BR) of Cue1p with Ubc7p. The U7BR is a unique E2-binding domain that includes three α-helices that interact extensively with the "backside" of Ubc7p. Residues essential for E2 binding are also required for activation of Ubc7p and for ERAD. We establish that the U7BR stimulates both RING-independent and RING-dependent ubiquitin transfer from Ubc7p. Moreover, the U7BR enhances ubiquitin-activating enzyme (E1)-mediated charging of Ubc7p with ubiquitin. This demonstrates that an essential component of E3 complexes can simultaneously bind to E2 and enhance its loading with ubiquitin. These findings provide mechanistic insights into how ubiquitination can be stimulated.

Allosteric activation of E2-RING finger-mediated ubiquitylation by a structurally defined specific E2-binding region of gp78.

The activity of RING finger ubiquitin ligases (E3) is dependent on their ability to facilitate transfer of ubiquitin from ubiquitin-conjugating enzymes (E2) to substrates. The G2BR domain within the E3 gp78 binds selectively and with high affinity to the E2 Ube2g2. Through structural and functional analyses, we determine that this occurs on a region of Ube2g2 distinct from binding sites for ubiquitin-activating enzyme (E1) and RING fingers. Binding to the G2BR results in conformational changes in Ube2g2 that affect ubiquitin loading. The Ube2g2:G2BR interaction also causes an approximately 50-fold increase in affinity between the E2 and RING finger. This results in markedly increased ubiquitylation by Ube2g2 and the gp78 RING finger. The significance of this G2BR effect is underscored by enhanced ubiquitylation observed when Ube2g2 is paired with other RING finger E3s. These findings uncover a mechanism whereby allosteric effects on an E2 enhance E2-RING finger interactions and, consequently, ubiquitylation.

The cytoplasmic Hsp70 chaperone machinery subjects misfolded and endoplasmic reticulum import-incompetent proteins to degradation via the ubiquitin-proteasome system.

The mechanism of protein quality control and elimination of misfolded proteins in the cytoplasm is poorly understood. We studied the involvement of cytoplasmic factors required for degradation of two endoplasmic reticulum (ER)-import-defective mutated derivatives of carboxypeptidase yscY (DeltassCPY* and DeltassCPY*-GFP) and also examined the requirements for degradation of the corresponding wild-type enzyme made ER-import incompetent by removal of its signal sequence (DeltassCPY). All these protein species are rapidly degraded via the ubiquitin-proteasome system. Degradation requires the ubiquitin-conjugating enzymes Ubc4p and Ubc5p, the cytoplasmic Hsp70 Ssa chaperone machinery, and the Hsp70 cochaperone Ydj1p. Neither the Hsp90 chaperones nor Hsp104 or the small heat-shock proteins Hsp26 and Hsp42 are involved in the degradation process. Elimination of a GFP fusion (GFP-cODC), containing the C-terminal 37 amino acids of ornithine decarboxylase (cODC) directing this enzyme to the proteasome, is independent of Ssa1p function. Fusion of DeltassCPY* to GFP-cODC to form DeltassCPY*-GFP-cODC reimposes a dependency on the Ssa1p chaperone for degradation. Evidently, the misfolded protein domain dictates the route of protein elimination. These data and our further results give evidence that the Ssa1p-Ydj1p machinery recognizes misfolded protein domains, keeps misfolded proteins soluble, solubilizes precipitated protein material, and escorts and delivers misfolded proteins in the ubiquitinated state to the proteasome for degradation.

A genome-wide screen identifies Yos9p as essential for ER-associated degradation of glycoproteins.

We undertook a growth-based screen exploiting the degradation of CTL*, a chimeric membrane-bound ERAD substrate derived from soluble lumenal CPY*. We screened the Saccharomyces cerevisiae genomic deletion library containing approximately 5000 viable strains for mutants defective in endoplasmic reticulum (ER) protein quality control and degradation (ERAD). Among the new gene products we identified Yos9p, an ER-localized protein previously involved in the processing of GPI anchored proteins. We show that deficiency in Yos9p affects the degradation only of glycosylated ERAD substrates. Degradation of non-glycosylated substrates is not affected in cells lacking Yos9p. We propose that Yos9p is a lectin or lectin-like protein involved in the quality control of N-glycosylated proteins. It may act sequentially or in concert with the ERAD lectin Htm1p/Mnl1p (EDEM) to prevent secretion of malfolded glycosylated proteins and deliver them to the cytosolic ubiquitin-proteasome machinery for elimination.

A Ubc7p-binding domain in Cue1p activates ER-associated protein degradation.

Cue1p is an N-terminally anchored endoplasmic reticulum (ER) protein essential for the activity of the two major yeast RING finger ubiquitin ligases (E3s) implicated in ER-associated degradation (ERAD). Cue1p contains a CUE domain, which for several proteins is known to bind ubiquitin. We now establish that the CUE domain is dispensable for ERAD of substrates of both Hrd1p and Doa10p and that the Cue1p transmembrane domain is similarly not required for degradation of the Hrd1p substrate CPY. Cue1p interacts with the ERAD E2 Ubc7p in vivo. We show that a discrete C-terminal Ubc7p binding region (U7BR) of Cue1p is required for ERAD and for Ubc7p-dependent ubiquitylation by Hrd1p in vitro. Strikingly, when Ubc7p is stabilized by direct anchoring to the ER membrane, the U7BR is sufficient to restore ERAD in cells lacking Cue1p. Thus, discrete E2 binding sites independent of ubiquitin ligase domains have the potential to activate ubiquitylation.

Ubiquitin ligases, critical mediators of endoplasmic reticulum-associated degradation.

Endoplasmic reticulum-associated degradation (ERAD) represents the primary means of quality control within the secretory pathway. Critical to this process are ubiquitin protein ligases (E3s) which, together with ubiquitin conjugating enzymes (E2s), mediate the ubiquitylation of proteins targeted for degradation from the ER. In this chapter we review our knowledge of both Saccharomyces cerevisiae and mammalian ERAD ubiquitin ligases. We focus on recent insights into these E3s, their associated proteins and potential mechanisms of action.

The ubiquitin ligase gp78 promotes sarcoma metastasis by targeting KAI1 for degradation.

Metastasis is the primary cause of mortality from cancer, but the mechanisms leading to metastasis are poorly understood. In particular, relatively little is known about metastasis in cancers of mesenchymal origins, which are known as sarcomas. Approximately ten proteins have been characterized as 'metastasis suppressors', but how these proteins function and are regulated is, in general, not well understood. Gp78 (also known as AMFR or RNF45) is a RING finger E3 ubiquitin ligase that is integral to the endoplasmic reticulum (ER) and involved in ER-associated degradation (ERAD) of diverse substrates. Here we report that expression of gp78 has a causal role in the metastasis of an aggressive human sarcoma and that this prometastatic activity requires the E3 activity of gp78. Further, gp78 associates with and targets the transmembrane metastasis suppressor, KAI1 (also known as CD82), for degradation. Suppression of gp78 increases KAI1 abundance and reduces the metastatic potential of tumor cells, an effect that is largely blocked by concomitant suppression of KAI1. An inverse relationship between these proteins was confirmed in a human sarcoma tissue microarray. Whereas most previous efforts have focused on genetic mechanisms for the loss of metastasis suppressor genes, our results provide new evidence for post-translational downregulation of a metastasis suppressor by its ubiquitin ligase, resulting in abrogation of its metastasis-suppressing effects.

Importance of carbohydrate positioning in the recognition of mutated CPY for ER-associated degradation.

In the endoplasmic reticulum (ER), N-linked glycans (N-glycans) function as signals to recruit the lectin chaperones involved in protein folding, quality control and ER-associated degradation. We undertook a systematic study of the four N-glycans of mutated carboxypeptidase yscY (CPY*) to determine whether there are positional differences between the glycans in ER-associated degradation. We constructed hypoglycosylated CPY* variants containing one, two or three N-glycans in various combinations and studied their degradation kinetics. We found that the four carbohydrate chains on CPY* are not equal in their signaling function: presence of the Asn368-linked glycan is necessary and sufficient for efficient degradation of CPY*. We also analysed the involvement of the ER lectins Htm1p and Cne1p (yeast calnexin) in the glycan-based recognition process with respect to number and position of N-glycans. We observed that Htm1p function depends on the presence of N-glycans in general but that there is no positional preference for a particular glycan. Cne1p, however, is selective with respect to substrate, and participates in the quality control only of some underglycosylated variants. For cases in which both lectins are involved, Cne1p and Htm1p play competing roles in targeting the substrate for degradation: loss of Cne1p accelerates degradation, whereas loss of Htm1p stabilizes the substrate.

For whom the bell tolls: protein quality control of the endoplasmic reticulum and the ubiquitin-proteasome connection.

The surveillance of the structural fidelity of the proteome is of utmost importance to all cells. The endoplasmic reticulum (ER) is the organelle responsible for proper folding and delivery of proteins to the secretory pathway. It contains a sophisticated protein proofreading and elimination mechanism. Failure of this machinery leads to disease and, finally, to cell death. Elimination of misfolded proteins requires retrograde transport across the ER membrane and depends on the central cytoplasmic proteolytic machinery involved in cellular regulation: the ubiquitin-proteasome system. The basics of this process as well as recent advances in the field are reviewed.

A genomic screen identifies Dsk2p and Rad23p as essential components of ER-associated degradation.

We developed a growth test to screen for yeast mutants defective in endoplasmic reticulum (ER) quality control and associated protein degradation (ERAD) using the membrane protein CTL*, a chimeric derivative of the classical ER degradation substrate CPY*. In a genomic screen of approximately 5,000 viable yeast deletion mutants, we identified genes necessary for ER quality control and degradation. Among the new gene products, we identified Dsk2p and Rad23p. We show that these two proteins are probably delivery factors for ubiquitinated ER substrates to the proteasome, following their removal from the membrane via the Cdc48-Ufd1-Npl4p complex. In contrast to the ERAD substrate CTG*, proteasomal degradation of a cytosolic CPY*-GFP fusion is not dependent on Dsk2p and Rad23p, indicating pathway specificity for both proteins. We propose that, in certain degradation pathways, Dsk2p, Rad23p and the trimeric Cdc48 complex function together in the delivery of ubiquitinated proteins to the proteasome, avoiding malfolded protein aggregates in the cytoplasm.

Comparative importance in vivo of conserved glutamate residues in the EX7E motif retaining glycosyltransferase Gpi3p, the UDP-GlcNAc-binding subunit of the first enzyme in glycosylphosphatidylinositol assembly.

Saccharomyces cerevisiae Gpi3p is the UDP-GlcNAc-binding and presumed catalytic subunit of the enzyme that forms GlcNAc-phosphatidylinositol in glycosylphosphatidylinositol biosynthesis. It is an essential protein with an EX7E motif that is conserved in four families of retaining glycosyltransferases. All Gpi3ps contain a cysteine residue four residues C-terminal to EX7E. To test their importance for Gpi3p function in vivo, Glu289 and 297 in the EX7E motif of S. cerevisiae Gpi3p, as well as Cys301, were altered by site-specific mutagenesis, and the mutant proteins tested for their ability to complement nonviable GPI3-deleted haploids. Gpi3p-C301A supported growth but membranes from C301A-expressing cells had low in vitro N-acetylglucosaminylphosphatidylinositol (GlcNAc-PI) synthetic activity. Haploids harboring Gpi3p-E289A proved viable, although slow growing but Gpi3-E297A did not support growth. The E289D and E297D mutants both supported growth at 25 degrees C, but, whereas the E289D strain grew at 37 degrees C, the E297D mutant did not. Membranes from E289D mutants had severely reduced in vitro GlcNAc-PI synthetic activity and E297D membranes had none. The mutation of the first Glu in the EX7E motif of Schizosaccharomyces pombe Gpi3p (Glu277) to Asp complemented the lethal null mutation in gpi3+ and supported growth at 37 degrees C, but the E285D mutant was nonviable. Our results suggest that the second Glu residue of the EX7E motif in Gpi3p is of greater importance than the first for function in vivo. Further, our findings do not support previous suggestions that the first Glu of an EX7E protein is the nucleophile and that Cys301 has an important role in UDP-GlcNAc binding by Gpi3ps.

Use of modular substrates demonstrates mechanistic diversity and reveals differences in chaperone requirement of ERAD.

The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.