Insulin-induced Gene Protein (INSIG)-dependent Sterol Regulation of Hmg2 Endoplasmic Reticulum-associated Degradation (ERAD) in Yeast

Insulin-induced gene proteins (INSIGs) function in control of cellular cholesterol. Mammalian INSIGs exert control by directly interacting with proteins containing sterol-sensing domains (SSDs) when sterol levels are elevated. Mammalian 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR) undergoes sterol-dependent, endoplasmic-reticulum (ER)-associated degradation (ERAD) that is mediated by INSIG interaction with the HMGR SSD. The yeast HMGR isozyme Hmg2 also undergoes feedback-regulated ERAD in response to the early pathway-derived isoprene gernanylgeranyl pyrophosphate (GGPP). Hmg2 has an SSD, and its degradation is controlled by the INSIG homologue Nsg1. However, yeast Nsg1 promotes Hmg2 stabilization by inhibiting GGPP-stimulated ERAD. We have proposed that the seemingly disparate INSIG functions can be unified by viewing INSIGs as sterol-dependent chaperones of SSD clients. Accordingly, we tested the role of sterols in the Nsg1 regulation of Hmg2. We found that both Nsg1-mediated stabilization of Hmg2 and the Nsg1-Hmg2 interaction required the early sterol lanosterol. Lowering lanosterol in the cell allowed GGPP-stimulated Hmg2 ERAD. Thus, Hmg2-regulated degradation is controlled by a two-signal logic; GGPP promotes degradation, and lanosterol inhibits degradation. These data reveal that the sterol dependence of INSIG-client interaction has been preserved for over 1 billion years. We propose that the INSIGs are a class of sterol-dependent chaperones that bind to SSD clients, thus harnessing ER quality control in the homeostasis of sterols.     

In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation

Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction. Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate. Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase. In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates. In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins. Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates. We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates. Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state. Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate.     

Ectopic RING activity at the ER membrane differentially impacts ERAD protein quality control pathways

Endoplasmic reticulum-associated degradation (ERAD) is a protein quality control pathway that ensures misfolded proteins are removed from the ER and destroyed. In ERAD, membrane and luminal substrates are ubiquitylated by ER-resident RING-type E3 ubiquitin ligases, retrotranslocated into the cytosol, and degraded by the proteasome. Overexpression of ERAD factors is frequently used in yeast and mammalian cells to study this process. Here, we analyze the impact of ERAD E3 overexpression on substrate turnover in yeast, where there are three ERAD E3 complexes (Doa10, Hrd1, and Asi1-3). Elevated Doa10 or Hrd1 (but not Asi1) RING activity at the ER membrane resulting from protein overexpression inhibits the degradation of specific Doa10 substrates. The ERAD E2 ubiquitin-conjugating enzyme Ubc6 becomes limiting under these conditions, and UBC6 overexpression restores Ubc6-mediated ERAD. Using a subset of the dominant-negative mutants, which contain the Doa10 RING domain but lack the E2-binding region, we show that they induce degradation of membrane tail-anchored Ubc6 independently of endogenous Doa10 and the other ERAD E3 complexes. This remains true even if the cells lack the Dfm1 rhomboid pseudoprotease, which is also a proposed retrotranslocon. Hence, rogue RING activity at the ER membrane elicits a highly specific off-pathway defect in the Doa10 pathway, and the data point to an additional ERAD E3-independent retrotranslocation mechanism.     

Mutations that affect vacuole biogenesis inhibit proliferation of the endoplasmic reticulum in Saccharomyces cerevisiae

In yeast, increased levels of the sterol biosynthetic enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase isozyme, Hmg1p, induce assembly of nuclear-associated ER membranes called karmellae. To identify additional genes involved in karmellae assembly, we screened temperature-sensitive mutants for karmellae assembly defects. Two independently isolated, temperature-sensitive strains that were also defective for karmellae biogenesis carried mutations in VPS16, a gene involved in vacuolar protein sorting. Karmellae biogenesis was defective in all 13 other vacuole biogenesis mutants tested, although the severity of the karmellae assembly defect varied depending on the particular mutation. The hypersensitivity of 14 vacuole biogenesis mutants to tunicamycin was well correlated with pronounced defects in karmellae assembly, suggesting that the karmellae assembly defect reflected alteration of ER structure or function. Consistent with this hypothesis, seven of eight mutations causing defects in secretion also affected karmellae assembly. However, the vacuole biogenesis mutants were able to proliferate their ER in response to Hmg2p, indicating that the mutants did not have a global defect in the process of ER biogenesis.     

Endoplasmic reticulum-associated degradation of the human type 2 iodothyronine deiodinase (D2) is mediated via an association between mammalian UBC7 and the carboxyl region of D2

The type 2 iodothyronine selenodeiodinase (D2) is an endoplasmic reticulum (ER)-resident selenoprotein that activates T4 to T3, playing a critical role in thyroid homeostasis. D2 has an approximately 45-min half-life due to selective ubiquitin-mediated ER-associated degradation (ERAD), a process of particular interest because it is accelerated by exposure to D2 substrates, T4 or rT3. The present in vitro binding studies indicate that glutathione-S-transferase (GST)-human D2 fusion proteins specifically associate with a mammalian homolog of the ubiquitin conjugase UBC7 (MmUBC7), with localization to amino acids 169-234 of D2. Coexpression of D2 with an inactive D2 mutant or a truncated version containing amino acids 169-234 stabilizes D2 half-life, supporting the importance of the carboxyl region of D2 for ERAD. Mammalian UBC6 (MmUBC6) does not directly associate with D2 but can associate with a complex containing UBC7 and D2. At the same time, functional studies in human embryonic kidney-293 cells indicate that D2 activity half-life and protein levels are stabilized only when inactive mutants of both UBC6 and UBC7 are overexpressed with D2, suggesting that redundancy may exist at the level of the E2 for both basal and substrate-accelerated D2 ERAD. In conclusion, D2 ERAD in human cells proceeds via an association between UBC7 and the carboxyl region of D2, a unique mechanism for the control of thyroid hormone activation.     

Mutant membrane protein of the budding yeast spindle pole body is targeted to the endoplasmic reticulum degradation pathway

Mutation of either the yeast MPS2 or the NDC1 gene leads to identical spindle pole body (SPB) duplication defects: The newly formed SPB is improperly inserted into the nuclear envelope (NE), preventing the cell from forming a bipolar mitotic spindle. We have previously shown that both MPS2 and NDC1 encode integral membrane proteins localized at the SPB. Here we show that CUE1, previously known to have a role in coupling ubiquitin conjugation to ER degradation, is an unusual dosage suppressor of mutations in MPS2 and NDC1. Cue1p has been shown to recruit the soluble ubiquitin-conjugating enzyme, Ubc7p, to the cytoplasmic face of the ER membrane where it can ubiquitinate its substrates and target them for degradation by the proteasome. Both mps2-1 and ndc1-1 are also suppressed by disruption of UBC7 or its partner, UBC6. The Mps2-1p mutant protein level is markedly reduced compared to wild-type Mps2p, and deletion of CUE1 restores the level of Mps2-1p to nearly wild-type levels. Our data indicate that Mps2p may be targeted for degradation by the ER quality control pathway.     

UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation

The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitin-conjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubc1 mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubc1ubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle.     

Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.

Ubiquitin-conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin-conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short-lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin-protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin-conjugation activity in stressed cells. Turnover of short-lived proteins and canavanyl-peptides but not of long-lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.     

Structural Basis for Ubiquitin Recognition by a Novel Domain from Human Phospholipase A2-activating Protein

Ubiquitin (Ub) is an essential modifier conserved in all eukaryotes from yeast to human. Phospholipase A₂-activating protein (PLAA), a mammalian homolog of yeast DOA1/UFD3, has been proposed to be able to bind with Ub, which plays important roles in endoplasmic reticulum-associated degradation, vesicle formation, and DNA damage response. We have identified a core domain from the PLAA family ubiquitin-binding region of human PLAA (residues 386–465, namely PFUC) that can bind Ub and elucidated its solution structure and Ub-binding mode by NMR approaches. The PFUC domain possesses equal population of two conformers in solution by cis/trans-isomerization, whereas the two isomers exhibit almost equivalent Ub binding abilities. This domain structure takes a novel fold consisting of four β-strands and two α-helices, and the Ub-binding site on PFUC locates in the surface of α2-helix, which is to some extent analogous to those of UBA, CUE, and UIM domains. This study provides structural basis and biochemical information for Ub recognition of the novel PFU domain from a PLAA family protein that may connect ubiquitination and degradation in endoplasmic reticulum-associated degradation.The eukaryotic secreted proteins are translocated into the endoplasmic reticulum after synthesis in cytosol. Misfolded or abnormally assembled proteins should be targeted for degradation through the endoplasmic reticulum-associated degradation (ERAD)³ pathway (1, 2). This pathway involves many molecular steps: unfolded protein response in the endoplasmic reticulum lumen, retrotranslocation back into the cytosol, ubiquitin (Ub) conjugation, delivery of ubiquitinated proteins to proteasome, and degradation of the substrates by proteases (3, 4).A yeast protein DOA1/UFD3 has been shown to bind to CDC48 by both indirect and direct ways (5–7), suggesting that DOA1 may be involved in ERAD. Evidence indicates that DOA1 directly competes with UFD2 at the same docking site on CDC48, which determines whether a substrate is multiubiquitinated and routed to the proteasome for degradation or deubiquitinated and released for other purposes (8). The direct interaction between DOA1 and Ub was suggested by recent studies (7, 9). Moreover, DOA1 also plays roles in the monoubiquitination of histone H2B and proliferating cell nuclear antigen (10) and in sorting ubiquitinated membrane proteins into multivesicular bodies (11).The mammalian homolog of DOA1 is called phospholipase A₂-activating protein (PLAA), which can bind to P97/VCP (a CDC48 homolog) with its C-terminal domain PUL (7). Having high sequence similarity (31% identity) with DOA1, PLAA is proposed to possess similar function of DOA1. Like DOA1, PLAA has an N-terminal WD40 domain with yet unknown function. The central region of PLAA contains a putative PLAA family ubiquitin-binding (PFU) domain, which is supposed to bind with Ub as observed in yeast DOA1 (7). Although the mechanism underlying the function of PLAA remains unclear, Ub binding of PLAA might be the central role that connects ubiquitination and degradation in ERAD. Thus, elucidating the molecular mechanism for specific binding of PLAA with Ub is prerequisite for understanding the function of PLAA as well as DOA1. To further understand the Ub-binding mechanism by which PLAA functions in ERAD pathway, we identified a small Ub-binding domain from human PLAA (12) and elucidated the domain structure and Ub-binding properties by NMR and mutagenesis approaches.     

INSIG: a broadly conserved transmembrane chaperone for sterol-sensing domain proteins

INSIGs are proteins that underlie sterol regulation of the mammalian proteins SCAP (SREBP cleavage activating protein) and HMG-CoA reductase (HMGR). The INSIGs perform distinct tasks in the regulation of these effectors: they promote ER retention of SCAP, but ubiquitin-mediated degradation of HMGR. Two questions that arise from the discovery and study of INSIGs are: how do they perform these distinct tasks, and how general are the actions of INSIGs in biology? We now show that the yeast INSIG homologs NSG1 and NSG2 function to control the stability of yeast Hmg2p, the HMGR isozyme that undergoes regulated ubiquitination. Yeast Nsgs inhibit degradation of Hmg2p in a highly specific manner, by directly interacting with the sterol-sensing domain (SSD)-containing transmembrane region. Nsg1p functions naturally to limit degradation of Hmg2p when both proteins are at native levels, indicating a long-standing functional interplay between these two classes of proteins. One way to unify the known, disparate actions of INSIGs is to view them as known adaptations of a chaperone dedicated to SSD-containing client proteins.     

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.     

Geranylgeranyl Pyrophosphate Is a Potent Regulator of HRD-dependent 3-Hydroxy-3-methylglutaryl-CoA Reductase Degradation in Yeast

3-Hydroxy-3-methylglutaryl (HMG)-CoA reductase (HMGR), the rate-limiting enzymes of sterol synthesis, undergoes feedback-regulated endoplasmic reticulum degradation in both mammals and yeast. The yeast Hmg2p isozyme is subject to ubiquitin-mediated endoplasmic reticulum degradation by the HRD pathway. We had previously shown that alterations in cellular levels of the 15-carbon sterol pathway intermediate farnesyl pyrophosphate (FPP) cause increased Hmg2p ubiquitination and degradation. We now present evidence that the FPP-derived, 20-carbon molecule geranylgeranyl pyrophosphate (GGPP) is a potent endogenous regulator of Hmg2p degradation. This work was launched by the unexpected observation that GGPP addition directly to living yeast cultures caused high potency and specific stimulation of Hmg2p degradation. This effect of GGPP was not recapitulated by FPP, GGOH, or related isoprenoids. GGPP-caused Hmg2p degradation met all the criteria for the previously characterized endogenous signal. The action of added GGPP did not require production of endogenous sterol molecules, indicating that it did not act by causing the build-up of an endogenous pathway signal. Manipulation of endogenous GGPP by several means showed that naturally made GGPP controls Hmg2p stability. Analysis of the action of GGPP indicated that the molecule works upstream of retrotranslocation and can directly alter the structure of Hmg2p. We propose that GGPP is the FPP-derived regulator of Hmg2p ubiquitination. Intriguingly, the sterol-dependent degradation of mammalian HMGR is similarly stimulated by the addition of GGOH to intact cells, implying that a dependence on 20-carbon geranylgeranyl signals may be a common conserved feature of HMGR regulation that may lead to highly specific therapeutic approaches for modulation of HMGR.     

Targeting of gp78 for ubiquitin-mediated proteasomal degradation by Hrd1: Cross-talk between E3s in the endoplasmic reticulum

There are an increasing number of ubiquitin ligases (E3s) implicated in endoplasmic reticulum (ER)-associated degradation (ERAD) in mammals. The two for which the greatest amount of information exists are the RING finger proteins gp78 and Hrd1, which are the structural orthologs of the yeast ERAD E3 Hrd1p. We now report that Hrd1, also known as synoviolin, targets gp78 for proteasomal degradation independent of the ubiquitin ligase activity of gp78, without evidence of a reciprocal effect. This degradation is observed in mouse embryonic fibroblasts lacking Hrd1, as well as with acute manipulation of Hrd1. The significance of this is underscored by the diminished level of a gp78-specific substrate, Insig-1, when Hrd1 expression is decreased and gp78 levels are consequently increased. These finding demonstrate a previously unappreciated level of complexity of the ubiquitin system in ERAD and have potentially important ramifications for processes where gp78 is implicated including regulation of lipid metabolism, metastasis, cystic fibrosis and neurodegenerative disorders.     

Arabidopsis ubiquitin conjugase UBC32 is an ERAD component that functions in brassinosteroid-mediated salt stress tolerance

Plants modify their growth and development to protect themselves from detrimental conditions by triggering a variety of signaling pathways, including the activation of the ubiquitin-mediated protein degradation pathway. Endoplasmic reticulum (ER)-associated protein degradation (ERAD) is an important aspect of the ubiquitin-proteasome system, but only a few of the active ERAD components have been reported in plants. Here, we report that the Arabidopsis thaliana ubiquitin-conjugating enzyme, UBC32, a stress-induced functional ubiquitin conjugation enzyme (E2) localized to the ER membrane, connects the ERAD process and brassinosteroid (BR)-mediated growth promotion and salt stress tolerance. In vivo data showed that UBC32 was a functional ERAD component that affected the stability of a known ERAD substrate, the barley (Hordeum vulgare) powdery mildew O (MLO) mutant MLO-12. UBC32 mutation caused the accumulation of bri1-5 and bri1-9, the mutant forms of the BR receptor, BRI1, and these mutant forms subsequently activated BR signal transduction. Further genetic and physiological data supported the contention that UBC32 plays a role in the BR-mediated salt stress response and that BR signaling is necessary for the plant to tolerate salt. Our data indicates a possible mechanism by which an ERAD component regulates the growth and stress response of plants.     

A Dominant Negative Mutant of Pma1 Interferes with the Folding of the Wild Type Enzyme

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1 , the gene coding for the plasma membrane H ⁺-ATPase, render misfolded proteins that are subjected to ERAD. A subset of misfolded PMA1 mutants exhibits a dominant negative effect on yeast growth since, when co-expressed with the wild type allele, both proteins are retained in the ER and degraded. We have used a PMA1 -D378T dominant lethal allele to analyse the mechanism underlying the retention of the wild type enzyme by the dominant negative mutant. A genetic screen was performed for isolation of intragenic suppressors of PMA1 -D378T allele. This analysis pointed to transmembrane helix 10 (TM10) as an important element in the establishment of the dominant lethality. Deletion of the TM10 was able to suppress not only the PMA1 -D378T but all the dominant lethal alleles tested. Biochemical analyses suggest that dominant lethal proteins obstruct, through TM10, the correct folding of the wild type enzyme leading to its retention and degradation by ERAD.     

Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast

Cystic fibrosis is the most widespread hereditary disease among the white population caused by different mutations of the apical membrane ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR). Its most common mutation, DeltaF508, leads to nearly complete degradation via endoplasmic reticulum-associated degradation (ERAD). Elucidation of the quality control and degradation mechanisms might give rise to new therapeutic approaches to cure this disease. In the yeast Saccharomyces cerevisiae, a variety of components of the protein quality control and degradation system have been identified. Nearly all of these components share homology with mammalian counterparts. We therefore used yeast mutants defective in the ERAD system to identify new components that are involved in human CFTR quality control and degradation. We show the role of the lectin Htm1p in the degradation process of CFTR. Complementation of the HTM1 deficiency in yeast cells by the mammalian orthologue EDEM underlines the necessity of this lectin for CFTR degradation and highlights the similarity of quality control and ERAD in yeast and mammals. Furthermore, degradation of CFTR requires the ubiquitin protein ligases Der3p/Hrd1p and Doa10p as well as the cytosolic trimeric Cdc48p-Ufd1p-Npl4p complex. These proteins also were found to be necessary for ERAD of a mutated yeast "relative" of CFTR, Pdr5(*)p.     

Dislocation of HMG-CoA Reductase and Insig-1, Two Polytopic Endoplasmic Reticulum Proteins,En Route to Proteasomal Degradation

The endoplasmic reticulum (ER) glycoprotein HMG-CoA reductase (HMGR) catalyzes the rate-limiting step in sterols biosynthesis. Mammalian HMGR is ubiquitinated and degraded by the proteasome when sterols accumulate in cells, representing the best example for metabolically controlled ER-associated degradation (ERAD). This regulated degradation involves the short-lived ER protein Insig-1. Here, we investigated the dislocation of these ERAD substrates to the cytosol en route to proteasomal degradation. We show that the tagged HMGR membrane region, HMG₃₅₀-HA, the endogenous HMGR, and Insig-1-Myc, all polytopic membrane proteins, dislocate to the cytosol as intact full-length polypeptides. Dislocation of HMG₃₅₀-HA and Insig-1-Myc requires metabolic energy and involves the AAA-ATPase p97/VCP. Sterols stimulate HMG₃₅₀-HA and HMGR release to the cytosol concurrent with removal of their N-glycan by cytosolic peptide:N-glycanase. Sterols neither accelerate dislocation nor stimulate deglycosylation of ubiquitination-defective HMG₃₅₀-HA^{(K89 + 248R)} mutant. Dislocation of HMG₃₅₀-HA depends on Insig-1-Myc, whose dislocation and degradation are sterol independent. Coimmunoprecipitation experiments demonstrate sterol-stimulated association between HMG₃₅₀-HA and Insig-1-Myc. Sterols do not enhance binding to Insig-1-Myc of HMG₃₅₀-HA mutated in its sterol-sensing domain or of HMG₃₅₀-HA^{(K89 + 248R)}. Wild-type HMG₃₅₀-HA and Insig-1-Myc coimmunoprecipitate from the soluble fraction only when both proteins were coexpressed in the same cell, indicating their encounter before or during dislocation, raising the possibility that they are dislocated as a tightly bound complex.     

Metabolically regulated endoplasmic reticulum-associated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase: evidence for requirement of a geranylgeranylated protein

In mammalian cells, the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes the rate-limiting step in the mevalonate pathway, is ubiquitylated and degraded by the 26 S proteasome when mevalonate-derived metabolites accumulate, representing a case of metabolically regulated endoplasmic reticulum-associated degradation (ERAD). Here, we studied which mevalonate-derived metabolites signal for HMGR degradation and the ERAD step(s) in which these metabolites are required. In HMGR-deficient UT-2 cells that stably express HMGal, a chimeric protein between β-galactosidase and the membrane region of HMGR, which is necessary and sufficient for the regulated ERAD, we tested inhibitors specific to different steps in the mevalonate pathway. We found that metabolites downstream of farnesyl pyrophosphate but upstream to lanosterol were highly effective in initiating ubiquitylation, dislocation, and degradation of HMGal. Similar results were observed for endogenous HMGR in cells that express this protein. Ubiquitylation, dislocation, and proteasomal degradation of HMGal were severely hampered when production of geranylgeranyl pyrophosphate was inhibited. Importantly, inhibition of protein geranylgeranylation markedly attenuated ubiquitylation and dislocation, implicating for the first time a geranylgeranylated protein(s) in the metabolically regulated ERAD of HMGR.     

ESCRT regulates surface expression of the Kir2.1 potassium channel

Protein quality control (PQC) is required to ensure cellular health. PQC is recognized for targeting the destruction of defective polypeptides, whereas regulated protein degradation mechanisms modulate the concentration of specific proteins in concert with physiological demands. For example, ion channel levels are physiologically regulated within tight limits, but a system-wide approach to define which degradative systems are involved is lacking. We focus on the Kir2.1 potassium channel because altered Kir2.1 levels lead to human disease and Kir2.1 restores growth on low-potassium medium in yeast mutated for endogenous potassium channels. Using this system, first we find that Kir2.1 is targeted for endoplasmic reticulum–associated degradation (ERAD). Next a synthetic gene array identifies nonessential genes that negatively regulate Kir2.1. The most prominent gene family that emerges from this effort encodes members of endosomal sorting complex required for transport (ESCRT). ERAD and ESCRT also mediate Kir2.1 degradation in human cells, with ESCRT playing a more prominent role. Thus multiple proteolytic pathways control Kir2.1 levels at the plasma membrane.     

Herp enhances ER-associated protein degradation by recruiting ubiquilins

ER-associated protein degradation (ERAD) is a protein quality control system of ER, which eliminates misfolded proteins by proteasome-dependent degradation and ensures export of only properly folded proteins from ER. Herp, an ER membrane protein upregulated by ER stress, is implicated in regulation of ERAD. In the present study, we show that Herp interacts with members of the ubiquilin family, which function as a shuttle factor to deliver ubiquitinated substrates to the proteasome for degradation. Knockdown of ubiquilin expression by small interfering RNA stabilized the ERAD substrate CD3δ, whereas it did not alter or increased degradation of non-ERAD substrates tested. CD3δ was stabilized by overexpressed Herp mutants which were capable of binding to ubiquilins but were impaired in ER membrane targeting by deletion of the transmembrane domain. Our data suggest that Herp binding to ubiquilin proteins plays an important role in the ERAD pathway and that ubiquilins are specifically involved in degradation of only a subset of ubiquitinated targets, including Herp-dependent ERAD substrates.