Hrd1p/Der3p is a membrane-anchored ubiquitin ligase required for ER-associated degradation.

In eukaryotes, endoplasmic reticulum-associated degradation (ERAD) functions in cellular quality control and regulation of normal ER-resident proteins. ERAD proceeds by the ubiquitin-proteasome pathway, in which the covalent attachment of ubiquitin to proteins targets them for proteasomal degradation. Ubiquitin-protein ligases (E3s) play a crucial role in this process by recognizing target proteins and initiating their ubiquitination. Here we show that Hrd1p, which is identical to Der3p, is an E3 for ERAD. Hrd1p is required for the degradation and ubiquitination of several ERAD substrates and physically associates with relevant ubiquitin-conjugating enzymes (E2s). A soluble Hrd1 fusion protein shows E3 activity in vitro - catalysing the ubiquitination of itself and test proteins. In this capacity, Hrd1p has an apparent preference for misfolded proteins. We also show that Hrd1p functions as an E3 in vivo, using only Ubc7p or Ubc1p to specifically program the ubiquitination of ERAD substrates.

Regulated degradation of HMG-CoA reductase, an integral membrane protein of the endoplasmic reticulum, in yeast.

Numerous integral membrane proteins are degraded in the mammalian ER. HMG-CoA reductase (HMG-R), a key enzyme in the mevalonate pathway by which isoprenoids and sterols are synthesized, is one substrate of ER degradation. The degradation of HMG-R is modulated by feedback signals from the mevalonate pathway. We investigated the role of regulated degradation of the two isozymes of HMG-R, Hmg1p and Hmg2p, in the physiology of Saccharomyces cerevisiae. Hmg1p was quite stable, whereas Hmg2p was rapidly degraded. Degradation of Hmg2p proceeded independently of vacuolar proteases or secretory traffic, indicating that Hmg2p degradation occurred at the ER. Hmg2p stability was strongly affected by modulation of the mevalonate pathway through pharmacological or genetic means. Decreased mevalonate pathway flux resulted in decreased degradation of Hmg2p. One signal for degradation of Hmg2p was a nonsterol, mevalonate-derived molecule produced before the synthesis of squalene. Genetic evidence indicated that a farnesylated protein may also be necessary for Hmg2p degradation. Studies with reporter genes demonstrated that the stability of each isozyme was determined by its noncatalytic NH2-terminal domain. Our data show that ER protein degradation is widely conserved among eukaryotes, and that feedback control of HMG-R degradation is an ancient paradigm of regulation.

Effects of changes in osmolality on the stability and function of cultured chromaffin cells and the possible role of osmotic forces in exocytosis.

Recent evidence indicates that osmotic forces may play a role in exocytosis. To examine this possibility and to investigate the osmotic properties of storage granules within cells, we investigated the effects of changes of osmolality on stability and function of cultured bovine chromaffin cells. Cell volume measurements indicated that the cells behaved as osmometers and that the intracellular osmolality rapidly equilibrated with the osmolality of the extracellular medium. Hyperosmotic solutions strongly inhibited nicotinic agonist-stimulated secretion but did not alter nicotinic agonist-stimulated Ca(2+) uptake. Hyperosmotic solutions also strongly inhibited elevated potassium- stimulated secretion but only weakly inhibited elevated K(+)-stimulated Ca(2+) uptake. Thus, hyperosmotic solutions inhibited secretion at a step after calcium entry. Cells exposed to 165 mOs(1) solutions did not lyse and retained their capacity to store and secrete catecholamine upon stimulation. Significant intracellular lysis of chromaffin granules occurred within cells exposed to lower osmolalities. In contrast, 75 percent of the catecholamine was released from granules from cultured cells or from fresh adrenal medulla incubated in vitro at 210 mOs. The data provide evidence for a role for osmotic forces in exocytosis and suggest that if osmotic stress of the granule occurs during exocytosis, then water influx into chromaffin granules increases granule volume by at least 70 percent. The results also indicate that the osmotic properties of the granules are altered upon homogenization and subcellular fractionation of the cells.

Regulation of HMG-CoA reductase degradation requires the P-type ATPase Cod1p/Spf1p.

The integral ER membrane protein HMG-CoA reductase (HMGR) is a key enzyme of the mevalonate pathway from which sterols and other essential molecules are produced. HMGR degradation occurs in the ER and is regulated by mevalonate-derived signals. Little is known about the mechanisms responsible for regulating HMGR degradation. The yeast Hmg2p isozyme of HMGR undergoes regulated degradation in a manner very similar to mammalian HMGR, allowing us to isolate mutants deficient in regulating Hmg2p stability. We call these mutants cod mutants for the control of HMG-CoA reductase degradation. With this screen, we have identified the first gene of this class, COD1, which encodes a P-type ATPase and is identical to SPF1. Our data suggested that Cod1p is a calcium transporter required for regulating Hmg2p degradation. This role for Cod1p is distinctly different from that of the well-characterized Ca(2+) P-type ATPase Pmr1p which is neither required for Hmg2p degradation nor its control. The identification of Cod1p is especially intriguing in light of the role Ca(2+) plays in the regulated degradation of mammalian HMGR.

Endoplasmic reticulum degradation requires lumen to cytosol signaling. Transmembrane control of Hrd1p by Hrd3p.

Endoplasmic reticulum (ER)-associated degradation (ERAD) is required for ubiquitin-mediated destruction of numerous proteins. ERAD occurs by processes on both sides of the ER membrane, including lumenal substrate scanning and cytosolic destruction by the proteasome. The ER resident membrane proteins Hrd1p and Hrd3p play central roles in ERAD. We show that these two proteins directly interact through the Hrd1p transmembrane domain, allowing Hrd1p stability by Hrd3p-dependent control of the Hrd1p RING-H2 domain activity. Rigorous reevaluation of Hrd1p topology demonstrated that the Hrd1p RING-H2 domain is located and functions in the cytosol. An engineered, completely lumenal, truncated version of Hrd3p functioned normally in both ERAD and Hrd1p stabilization, indicating that the lumenal domain of Hrd3p regulates the cytosolic Hrd1p RING-H2 domain by signaling through the Hrd1p transmembrane domain. Additionally, we identified a lumenal region of Hrd3p dispensable for regulation of Hrd1p stability, but absolutely required for normal ERAD. Our studies show that Hrd1p and Hrd3p form a stoichiometric complex with ERAD determinants in both the lumen and the cytosol. The HRD complex engages in lumen to cytosol communication required for regulation of Hrd1p stability and the coordination of ERAD events on both sides of the ER membrane.

Cholesterol homeostasis: ESCAPe from the ER.

Transcriptional regulation and membrane traffic have traditionally been quite separate fields of biology, but they have been brought under the same roof by recent advances in understanding the cellular control of cholesterol metabolism.

ER stress response: getting the UPR hand on misfolded proteins.

Unfolded proteins are constantly delivered to the ER lumen, where they must be removed by folding or degradation. Recent studies show that the 'unfolded protein response' controls essentially all aspects of ER function, coordinating these two fates for misfolded proteins in a process necessary for normal cell life.

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.

Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the agents of HMG-R degradation but not the regulators of that degradation.

HRD4/NPL4 is required for the proteasomal processing of ubiquitinated ER proteins.

We isolated a temperature-sensitive mutant, hrd4-1, deficient in ER-associated degradation (ERAD). The HRD4 gene was identical to NPL4, a gene previously implicated in nuclear transport. Using a diverse set of substrates and direct ubiquitination assays, our analysis revealed that HRD4/NPL4 is required for a poorly characterized step in ERAD after ubiquitination of target proteins but before their recognition by the 26S proteasome. Our data indicate that this lack of proteasomal processing of ubiquitinated proteins constitutes the primary defect in hrd4/npl4 mutant cells and explains the diverse set of hrd4/npl4 phenotypes. We also found that each member of the Cdc48p-Ufd1p-Npl4p complex is individually required for ERAD.

Stereospecific binding of 3H-phencyclidine in brain membranes.

Phencyclidine (PCP) displaceable binding of 3H-PCP to glass-fiber filters was eliminated and total binding markedly reduced by initial treatment of the discs with 0.05% polyethyleneimine. Assessed with treated filters, unlabeled PCP displaced 3H-PCP in both rat and pigeon brain membranes with an EC50 of 1 microM. Of similar high inhibitory potency were dextrorphan, levorphanol, SKF 10047 and ketamine, while morphine, naloxone and etorphine had EC50 values higher then 1 mM. Using the dissociative anesthetic dexoxadrol and its inactive isomer levoxadrol as displacing agents, stereospecific binding of 3H-PCP was obtained in rat and pigeon brain membranes. The markedly higher potency of dexoxadrol, relative to levoxadrol, in displacing bound 3H-PCP is compatible with behavioral data for these enantiomers. However, they were equipotent in displacing 3H-PCP bound to glass-fiber filters in the absence of tissue. Heat denaturation, but not freezing, abolished stereospecific binding of 3H-PCP, which was also absent in rat liver membranes. The stereospecific binding component in brain displayed biphasic saturability at 60-70 nM and 300-400 nM, respectively.

Aggregation behavior of lipid IVA in aqueous solutions at physiological pH. 1: Simple buffer solutions.

We have investigated the aggregation behaviour of lipid IVA (a bioactive precursor of lipid A and the lipid anchor of lipopolysaccharide) in aqueous solutions in the physiological pH range using dynamic light scattering, nuclear magnetic resonance, fluorescence, surface pressure, electron microscopy and force field simulation studies. The sonication of lipid IVA in PBS, Tris and Hepes produces vesicles which are stable in the concentration range of 10(-3) - 10(-7) M, possibly even at lower concentrations. The vesicle size is not sensitive to the nature of the buffer, only to the pH and to some extent to the ionic strength. The long time stability of the small unilamellar vesicles as well as the structureless 1H-NMR spectra might be attributed to a rigid surface structure. This structure is also supported by the simulation studies. We have tentatively proposed a coexistence of micelles and/or other aggregates with the bilayered vesicles at higher lipid concentrations in order to explain some of the experimental observations.

Genetic analysis of hydroxymethylglutaryl-coenzyme A reductase regulated degradation.

Hydroxymethylglutaryl-coenzyme A reductase degradation occurs in the endoplasmic reticulum, and is regulated by the mevalonate pathway. In order to discover the molecules that mediate the degradation process and its control, we conducted a genetic analysis of the degradation of the yeast Hmg2p isozyme of hydroxymethylglutaryl-coenzyme A reductase. Hmg2p degradation occurs by the action of HRD genes that direct Hmg2p to the ubiquitin-proteasome pathway. Regulation of HRD-dependent Hmg2p degradation appears to occur by the action of a separate set of CRD genes.

Macrophage catabolism of lipid A is regulated by endotoxin stimulation.

Lipopolysaccharide (LPS) is a Gram-negative bacterial glycolipid that is believed to cause, by virtue of its stimulatory actions on macrophages and other eukaryotic cells, the life-threatening symptoms associated with Gram-negative infections. Macrophages both respond to and catabolically deactivate LPS. The lipid A moiety of LPS is responsible for the stimulatory actions of LPS on macrophages. We have previously developed methods employing a radiolabeled bioactive lipid A precursor, 4'-32P-lipid IVA, to study the interaction of this class of lipids with animal cells (Hampton, R. Y., Golenbock, D. T., and Raetz, C. R. H. (1988). J. Biol. Chem. 263, 14802-14807). In the current work, we have examined the uptake and catabolism of 4'-32P-lipid IVA by the RAW 264.7 cell line in serum-containing medium at physiological temperatures and have studied the effect of LPS stimulation on the ability of these cells to catabolize lipid IVA. RAW 264.7 macrophage-like cells avidly take up 4'-32P-lipid IVA under cell culture conditions at nanomolar concentrations. Uptake of lipid IVA was accompanied by lysosomal dephosphorylation of a fraction of the lipid to yield 4'-monophosphoryl lipid IVA. Chemically generated 4'-monophosphoryl lipid IVA was found to be substantially less bioactive than lipid IVA in the RAW cell, indicating that this catabolic dephosphorylation results in detoxification. In uptake experiments of 3-4 h duration, all metabolism of lipid IVA is blocked by ligands of the macrophage scavenger receptor. In longer experiments (24 h), both scavenger receptor-dependent and -independent uptake are responsible for the lysosomal catabolism of lipid IVA. Preincubation of RAW 264.7 cells with LPS caused dose-dependent inhibition of lipid IVA dephosphorylation. Sufficient LPS stimulation resulted in essentially complete inhibition of lipid IVA catabolism in both short- and long-term uptake experiments. This effect occurred at physiologically relevant concentrations of LPS (IC50 less than 1 ng/ml), and our data indicate that LPS-induced blockade of lipid IVA catabolism was due to the resultant physiological stimulation of the cells, and not inhibition of dephosphorylation by competition for uptake or enzymatic sites or by simple sequestration of labeled lipid IVA by LPS aggregates. We suggest that in the macrophage, LPS can modulate its own catabolism by virtue of its pharmacological properties. This effect of LPS could play a role in LPS pathophysiology as well as in macrophage biology.

A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes.

Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation. To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast. We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast. This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general. Rather, the FPP-derived signal specifically modulates Hmg2p stability. In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation. Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes.

A 'distributed degron' allows regulated entry into the ER degradation pathway.

Protein degradation is employed in both regulation and quality control. Regulated degradation of specific proteins is often mediated by discrete regions of primary sequence known as degrons, whereas protein quality control involves recognition of structural features common to damaged or misfolded proteins, rather than specific features of an individual protein. The yeast HMG-CoA reductase isozyme Hmg2p undergoes stringently regulated degradation by machinery that is also required for ER quality control. The 523 residue N-terminal transmembrane domain of Hmg2p is necessary and sufficient for regulated degradation. To understand how Hmg2p undergoes regulated degradation by the ER quality control pathway, we analyzed over 300 mutants of Hmg2p. Regulated degradation of Hmg2p requires information distributed over the entire transmembrane domain. Accordingly, we refer to this determinant as a 'distributed' degron, which has functional aspects consistent with both regulation and quality control. The Hmg2p degron functions in the specific, regulated degradation of Hmg2p and can impart regulated degradation to fusion proteins. However, its recognition is based on dispersed structural features rather than primary sequence motifs. This mode of targeting has important consequences both for the prediction of degradation substrates and as a potential therapeutic strategy for targeted protein degradation using endogenous degradation pathways.

Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase.

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.