A novel mammalian endoplasmic reticulum ubiquitin ligase homologous to the yeast Hrd1

The yeast hHrd1 is a ubiquitin-protein ligase (E3) involved in ER-associated degradation. It was originally identified by genetic methods as an E3 of the yeast cholesterol biosynthetic enzyme HMG-CoA reductase (HMGR). We report the identification and cloning of a human homologue of Hrd1 (hHrd1). Immunofluorescence imaging confirms that the endogenous hHrd1 resides in the ER and in vitro assay demonstrates that it has a ubiquitin-ligase activity. However, the homology between the human and yeast Hrd1 is limited to the N-terminal domain of the proteins, and hHrd1 does not appear to be involved in the 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.     

Voa1p functions in V-ATPase assembly in the yeast endoplasmic reticulum

The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V₁ sector catalyzes ATP hydrolysis and the V₀ sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V₀ assembly. We have discovered a fifth V₀ assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V₀ assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V₀ assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V₀–Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V₀ subunits c, c′, and c″. On assembly of the remaining three V₀ subunits (a, d, and e) into the V₀ complex, Voa1p dissociates from the now fully assembled V₀–Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V₀ assembly in the ER, but then it dissociates before exit of the V₀–Vma21p complex from the ER for transport to the Golgi compartment.     

A role for the protease-sensitive loop region of Shiga-like toxin 1 in the retrotranslocation of its A1 domain from the endoplasmic reticulum lumen

Shiga-like toxin I (Slt-I) is a ribosome-inactivating protein that undergoes retrograde transport to the endoplasmic reticulum to exert its cytotoxic effect on eukaryotic cells. Its catalytically active A(1) domain subsequently migrates from the endoplasmic reticulum (ER) lumen to the cytoplasm. To study this final retrotranslocation event, a suicide assay was developed based on the cytoplasmic expression and ER-targeting of the cytotoxic Slt-I A(1) fragment in Saccharomyces cerevisiae. Expression of the Slt-I A(1) domain (residues 1-251) with and without an ER-targeting sequence was lethal to the host and demonstrated that this domain can efficiently migrate from the ER compartment to the cytosol. Deletion analyses revealed that residues 1-239 represent the minimal A(1) segment displaying full enzymatic activity. This fragment, however, accumulates in the ER lumen when directed to this compartment. The addition of residues 240-251 restores the translocation property of the A(1) chain in yeast. However, single mutations within this region do not significantly alter this function in the context of the 251-residue long A(1) domain or affect the toxicity of the resulting Slt-I variants toward Vero cells in the context of the holotoxin. Since this mechanism of retrotranslocation is common to other protein toxins lacking a peptide motif similar in sequence to residues 240-251, the present results suggest that the ER export mechanism may involve the recognition of a more universal structural element, such as a misfolded or altered peptide domain localized at the C terminus of the A(1) chain (residues 240-251) rather than a unique ER export signal sequence.     

Ssh1p determines the translocation and dislocation capacities of the yeast endoplasmic reticulum.

Sec61p is required both for protein translocation and dislocation across the membrane of the endoplasmic reticulum (ER). However, the cellular role of the Sec61p homolog Ssh1p has not been clearly defined. We show that deltassh1 mutant cells have strong defects in both SRP-dependent and -independent translocation. Moreover, these cells were also found to be induced for the unfolded protein response and to be defective in dislocation of a misfolded ER protein. In addition, deltassh1 mutant cells rapidly became respiratory deficient. The other defects discussed above were suppressed in the respiratory-deficient state or under conditions where the rate of polypeptide translation was artificially reduced. These data identify Ssh1p as a component of a second, functionally distinct translocon in the yeast ER membrane.     

Crystallization of the B chain of Shiga-like toxin I from Escherichia coli

Shiga-like toxin I (SLT-I) is produced by several pathogenic strains of Escherichia coli associated with diarrheal disease. The toxin consists of an A chain, which attacks eukaryotic ribosomes, inhibiting protein synthesis, and multiple copies of a 69 amino acid B chain. The B subunit mediates cell binding and uptake through its interactions with cell surface carbohydrate moieties. Here we report that the B chain has been crystallized in a form suitable for high-resolution X-ray analysis. The space group is P2(1)2(1)2(1), with a = 56.2 A, b = 59.9 A and c = 102.5 A. A rotation function using three-dimensional diffraction data suggests that the asymmetric unit is a tetramer.     

Retrotranslocation of a viral A/B toxin from the yeast endoplasmic reticulum is independent of ubiquitination and ERAD

K28 is a viral A/B toxin that traverses eukaryotic cells by endocytosis and retrograde transport through the secretory pathway. Here we show that toxin retrotranslocation from the endoplasmic reticulum (ER) requires Kar2p/BiP, Pdi1p, Scj1p, Jem1p, and proper maintenance of Ca(2+) homeostasis. Neither cytosolic chaperones nor Cdc48p/Ufd1p/Npl4p complex components or proteasome activity are required for ER exit, indicating that K28 retrotranslocation is mechanistically different from classical ER-associated protein degradation (ERAD). We demonstrate that K28 exits the ER in a heterodimeric but unfolded conformation and dissociates into its subunits as it emerges into the cytosol where beta is ubiquitinated and degraded. ER export and in vivo toxicity were not affected in a lysine-free K28 variant nor under conditions when ubiquitination and proteasome activity was blocked. In contrast, toxin uptake from the plasma membrane required Ubc4p (E2) and Rsp5p (E3) and intoxicated ubc4 and rsp5 mutants accumulate K28 at the cell surface incapable of toxin internalization. We propose a model in which ubiquitination is involved in the endocytic pathway of the toxin, while ER-to-cytosol retrotranslocation is independent of ubiquitination, ERAD and proteasome activity.     

Sls1p stimulates Sec63p-mediated activation of Kar2p in a conformation-dependent manner in the yeast endoplasmic reticulum

We previously characterized the SLS1 gene in the yeast Yarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903-30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between DeltaScsls1 and translocation-deficient kar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding of ScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.     

Membrane topology and function of Der3/Hrd1p as a ubiquitin-protein ligase (E3) involved in endoplasmic reticulum degradation

The endoplasmic reticulum contains a protein quality control system that discovers malfolded or unassembled secretory proteins and subjects them to degradation in the cytosol. This requires retrograde transport of the respective proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and degradation. Ubiquitination of mutated and malfolded proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for degradation. Here we present the topology of the previously discovered RING-H2 finger protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-protein ligase (E3) of the endoplasmic reticulum-associated protein degradation process.     

The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.     

A protein complex containing Epo1p anchors the cortical endoplasmic reticulum to the yeast bud tip

The cortical endoplasmic reticulum (cER) of yeast underlies the plasma membrane (PM) at specific contact sites to enable a direct transfer of information and material between both organelles. During budding, directed movement of cER to the young bud followed by subsequent anchorage at its tip ensures the faithful inheritance of this organelle. The ER membrane protein Scs2p tethers the cER to the PM and to the bud tip through so far unknown receptors. We characterize Epo1p as a novel member of the polarisome that interacts with Scs2p exclusively at the cell tip during bud growth and show that Epo1p binds simultaneously to the Cdc42p guanosine triphosphatase–activating protein Bem3p. Deletion of EPO1 or deletion of BEM3 in a polarisome-deficient strain reduces the amount of cER at the tip. This analysis therefore identifies Epo1p as a novel and important component of the polarisome that promotes cER tethering at sites of polarized growth.     

Nuclear pore complex integrity requires Lnp1, a regulator of cortical endoplasmic reticulum

The nuclear envelope (NE) and endoplasmic reticulum (ER) are components of the same contiguous membrane system and yet have distinct cellular functions. Mounting evidence suggests roles for some ER proteins in the NE for proper nuclear pore complex (NPC) structure and function. In this study, we identify a NE role in Saccharomyces cerevisiae for Lnp1 and Sey1, proteins required for proper cortical ER formation. Both lnp1Δ and sey1Δ mutants exhibit synthetic genetic interactions with mutants in genes encoding key NPC structural components. Both Lnp1 and Sey1 physically associate with other ER components that have established NPC roles, including Rtn1, Yop1, Pom33, and Per33. Of interest, lnp1Δ rtn1Δ mutants but not rtn1Δ sey1Δ mutants exhibit defects in NPC distribution. Furthermore, the essential NPC assembly factor Ndc1 has altered interactions in the absence of Sey1. Lnp1 dimerizes in vitro via its C-terminal zinc finger motif, a property that is required for proper ER structure but not NPC integrity. These findings suggest that Lnp1''s role in NPC integrity is separable from functions in the ER and is linked to Ndc1 and Rtn1 interactions.     

Integration of membrane proteins into the endoplasmic reticulum requires GTP

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.     

A RING-H2 finger motif is essential for the function of Der3/Hrd1 in endoplasmic reticulum associated protein degradation in the yeast Saccharomyces cerevisiae

Der3/Hrd1p is a protein required for proper degradation of misfolded soluble and integral membrane proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N-terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3deltaRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the protein in ER degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3(C399S) protein maintains the correct localization and topology of the wild-type protein, however, is not able to support the degradation of soluble and integral membrane proteins. This point mutation altering the RING-H2 motif behaves as a dominant allele especially when overexpressed from a 2mu plasmid by this increasing the half-life of CPY* more than 6-fold when compared with a wild-type strain. Furthermore coexpression of der3(C399S) with the wild-type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61-2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3(C399S) mutation. These results could be explained by a competition between wild-type and mutant Der3 protein for the interaction with some other component of the ER degradation pathway, probably Hrd3p.     

Sac1p mediates the adenosine triphosphate transport into yeast endoplasmic reticulum that is required for protein translocation

Protein translocation into the yeast endoplasmic reticulum requires the transport of ATP into the lumen of this organelle. Microsomal ATP transport activity was reconstituted into proteoliposomes to characterize and identify the transporter protein. A polypeptide was purified whose partial amino acid sequence demonstrated its identity to the product of the SAC1 gene. Accordingly, microsomal membranes isolated from strains harboring a deletion in the SAC1 gene (sac1 delta) were found to be deficient in ATP-transporting activity as well as severely compromised in their ability to translocate nascent prepro- alpha-factor and preprocarboxypeptidase Y. Proteins isolated from the microsomal membranes of a sac1 delta strain were incapable of stimulating ATP transport when reconstituted into the in vitro assay system. When immunopurified to homogeneity and incorporated into artificial lipid vesicles, Sac1p was shown to reconstitute ATP transport activity. Consistent with the requirement for ATP in the lumen of the ER to achieve the correct folding of secretory proteins, the sac1 delta strain was shown to have a severe defect in transport of procarboxypeptidase Y out of the ER and into the Golgi complex in vivo. The collective data indicate an intimate role for Sac1p in the transport of ATP into the ER lumen.     

Sulfatase modifying factor 1 trafficking through the cells: from endoplasmic reticulum to the endoplasmic reticulum

Sulfatase modifying factor 1 (SUMF1) is the gene mutated in multiple sulfatase deficiency (MSD) that encodes the formylglycine-generating enzyme, an essential activator of all the sulfatases. SUMF1 is a glycosylated enzyme that is resident in the endoplasmic reticulum (ER), although it is also secreted. Here, we demonstrate that upon secretion, SUMF1 can be taken up from the medium by several cell lines. Furthermore, the in vivo engineering of mice liver to produce SUMF1 shows its secretion into the blood serum and its uptake into different tissues. Additionally, we show that non-glycosylated forms of SUMF1 can still be secreted, while only the glycosylated SUMF1 enters cells, via a receptor-mediated mechanism. Surprisingly, following its uptake, SUMF1 shuttles from the plasma membrane to the ER, a route that has to date only been well characterized for some of the toxins. Remarkably, once taken up and relocalized into the ER, SUMF1 is still active, enhancing the sulfatase activities in both cultured cells and mice tissues.     

A mammalian homologue of SLY1 a yeast gene required for transport from endoplasmic reticulum to Golgi

We characterized rat cDNAs that predict a protein, r-Slyl, which is similar to SLY1, a yeast protein that plays a critical role in endoplasmic reticulum to Golgi apparatus vesicle trafficking. The r-Slyl gene is expressed in all tissues examined     

Concentrative export from the endoplasmic reticulum of the gamma-aminobutyric acid transporter 1 requires binding to SEC24D

Re-uptake of gamma-aminobutyric acid (GABA) into presynaptic specializations is mediated by the GABA transporter 1 (GAT1), a member of the SLC6 gene family. Here, we show that a motif in the COOH terminus of GAT1 ((566)RL(567)), which is conserved in SLC6 family members, is a binding site for the COPII coat component Sec24D. We also identified residues in Sec24D ((733)DD(734)) that are required to support the interaction with GAT1 and two additional family members, i.e. the transporters for serotonin and dopamine. We used three strategies to prevent recruitment of Sec24D to GAT1: knock-down of Sec24D by RNA interference, overexpression of Sec24D-VN (replacement of (733)DD(734) by (733)VN(734)), and mutation of (566)RL(567) to (566)AS(567) (GAT1-RL/AS). In each instance, endoplasmic reticulum (ER) export of GAT1 was impaired: in the absence of Sec24D or upon coexpression of dominant negative Sec24D-VN, GAT1 failed to undergo concentrative ER export; GAT1-RL/AS also accumulated in the ER and exerted a dominant negative effect on cell surface targeting of wild type GAT1. Our observations show that concentrative ER-export is contingent on a direct interaction of GAT1 with Sec24D; this also provides a mechanistic explanation for the finding that oligomeric assembly of transporters is required for their ER export: transporter oligomerization supports efficient recruitment of COPII components.     

Analysis of membrane topology and identification of essential residues for the yeast endoplasmic reticulum inositol acyltransferase Gwt1p

Glycosylphosphatidylinositol (GPI) is a post-translational modification that anchors cell surface proteins to the plasma membrane, and GPI modifications occur in all eukaryotes. Biosynthesis of GPI starts on the cytoplasmic face of the endoplasmic reticulum (ER) membrane, and GPI precursors flip from the cytoplasmic side to the luminal side of the ER, where biosynthesis of GPI precursors is completed. Gwt1p and PIG-W are inositol acyltransferases that transfer fatty acyl chains to the inositol moiety of GPI precursors in yeast and mammalian cells, respectively. To ascertain whether flipping across the ER membrane occurs before or after inositol acylation of GPI precursors, we identified essential residues of PIG-W and Gwt1p and determined the membrane topology of Gwt1p. Guided by algorithm-based predictions of membrane topology, we experimentally identified 13 transmembrane domains in Gwt1p. We found that Gwt1p, PIG-W, and their orthologs shared four conserved regions and that these four regions in Gwt1p faced the luminal side of the ER membrane. Moreover, essential residues of Gwt1p and PIG-W faced the ER lumen or were near the luminal edge of transmembrane domains. The membrane topology of Gwt1p suggested that inositol acylation occurred on the luminal side of the ER membrane. Rather than stimulate flipping of the GPI precursor across the ER membrane, inositol acylation of GPI precursors may anchor the precursors to the luminal side of the ER membrane, preventing flip-flops.