Oxidative Activity of Yeast Ero1p on Protein Disulfide Isomerase and Related Oxidoreductases of the Endoplasmic Reticulum

The sulfhydryl oxidase Ero1 oxidizes protein disulfide isomerase (PDI), which in turn catalyzes disulfide formation in proteins folding in the endoplasmic reticulum (ER). The extent to which other members of the PDI family are oxidized by Ero1 and thus contribute to net disulfide formation in the ER has been an open question. The yeast ER contains four PDI family proteins with at least one potential redox-active cysteine pair. We monitored the direct oxidation of each redox-active site in these proteins by yeast Ero1p in vitro. In this study, we found that the Pdi1p amino-terminal domain was oxidized most rapidly compared with the other oxidoreductase active sites tested, including the Pdi1p carboxyl-terminal domain. This observation is consistent with experiments conducted in yeast cells. In particular, the amino-terminal domain of Pdi1p preferentially formed mixed disulfides with Ero1p in vivo, and we observed synthetic lethality between a temperature-sensitive Ero1p variant and mutant Pdi1p lacking the amino-terminal active-site disulfide. Thus, the amino-terminal domain of yeast Pdi1p is on a preferred pathway for oxidizing the ER thiol pool. Overall, our results provide a rank order for the tendency of yeast ER oxidoreductases to acquire disulfides from Ero1p.     

Yet1p and Yet3p,the Yeast Homologs of BAP29 and BAP31, Interact with the Endoplasmic Reticulum Translocation Apparatus and Are Required for Inositol Prototrophy

The mammalian B-cell receptor-associated proteins of 29 and 31 kDa (BAP29 and BAP31) are conserved integral membrane proteins that have reported roles in endoplasmic reticulum (ER) quality control, ER export of secretory cargo, and programmed cell death. In this study we investigated the yeast homologs of BAP29 and BAP31, known as Yet1p and Yet3p, to gain insight on cellular function. We found that Yet1p forms a complex with Yet3p (Yet complex) and that complex assembly was important for subunit stability and proper ER localization. The Yet complex was not efficiently packaged into ER-derived COPII vesicles and therefore does not appear to act as an ER export receptor. Instead, a fraction of the Yet complex was detected in association with the ER translocation apparatus (Sec complex). Specific mutations in the Sec complex or Yet complex influenced these interactions. Moreover, associations between the Yet complex and Sec complex were increased by ER stress and diminished when protein translocation substrates were depleted. Surprisingly, yet1Δ and yet3Δ mutant strains displayed inositol starvation-related growth defects. In accord with the biochemical data, these growth defects were exacerbated by a combination of certain mutations in the Sec complex with yet1Δ or yet3Δ mutations. We propose a model for the Yet-Sec complex interaction that places Yet1p and Yet3p at the translocation pore to manage biogenesis of specific transmembrane secretory proteins.     

Novel Thioredoxin-related Transmembrane Protein TMX4 Has Reductase Activity

In the endoplasmic reticulum (ER), a number of thioredoxin (Trx) superfamily proteins are present to enable correct disulfide bond formation of secretory and membrane proteins via Trx-like domains. Here, we identified a novel transmembrane Trx-like protein 4 (TMX4), in the ER of mammalian cells. TMX4, a type I transmembrane protein, was localized to the ER and possessed a Trx-like domain that faced the ER lumen. A maleimide alkylation assay showed that a catalytic CXXC motif in the TMX4 Trx-like domain underwent changes in its redox state depending on cellular redox conditions, and, in the normal state, most of the endogenous TMX4 existed in the oxidized form. Using a purified recombinant protein containing the Trx-like domain of TMX4 (TMX4-Trx), we confirmed that this domain had reductase activity in vitro. The redox potential of this domain (−171.5 mV; 30 °C at pH 7.0) indicated that TMX4 could work as a reductase in the environment of the ER. TMX4 had no effect on the acceleration of ER-associated degradation. Because TMX4 interacted with calnexin and ERp57 by co-immunoprecipitation assay, the role of TMX4 may be to enable protein folding in cooperation with these proteins consisting of folding complex in the ER.     

The Vacuole Import and Degradation Pathway Utilizes Early Steps of Endocytosis and Actin Polymerization to Deliver Cargo Proteins to the Vacuole for Degradation

When glucose is added to yeast cells that are starved for 3 days, fructose-1,6-bisphosphatase (FBPase) and malate dehydrogenase 2 are degraded in the vacuole via the vacuole import and degradation (Vid) pathway. In this study, we examined the distribution of FBPase at the ultrastructural level. FBPase was observed in areas close to the plasma membrane and in cytoplasmic structures that are heterogeneous in size and density. We have isolated these intracellular structures that contain FBPase, the Vid vesicle marker Vid24p, and the endosomal marker Pep12p. They appeared irregular in size and shape. In yeast, actin polymerization plays an important role in early steps of endocytosis. Mutants that affect actin polymerization inhibited FBPase degradation, suggesting that actin polymerization is important for FBPase degradation. Both FBPase and malate dehydrogenase 2 were associated with actin patches. Vid vesicle proteins such as Vid24p or Sec28p were also at actin patches, although they dissociated from these structures at later time points. We propose that Vid24p and Sec28p are present at actin patches during glucose starvation. Cargo proteins arrive at these sites following the addition of glucose, and the endocytic vesicles then pinch off from the plasma membrane. Following the fusion of endosomes with the vacuole, cargo proteins are then degraded in the vacuole.     

Conserved Loop Cysteines of Vitamin K Epoxide Reductase Complex Subunit 1-like 1 (VKORC1L1) Are Involved in Its Active Site Regeneration

Vitamin K epoxide reductase complex subunit 1 (VKORC1) reduces vitamin K epoxide in the vitamin K cycle for post-translational modification of proteins that are involved in a variety of biological functions. However, the physiological function of VKORC1-like 1 (VKORC1L1), a paralogous enzyme sharing about 50% protein identity with VKORC1, is unknown. Here we determined the structural and functional differences of these two enzymes using fluorescence protease protection (FPP) assay and an in vivo cell-based activity assay. We show that in vivo VKORC1L1 reduces vitamin K epoxide to support vitamin K-dependent carboxylation as efficiently as does VKORC1. However, FPP assays show that unlike VKORC1, VKORC1L1 is a four-transmembrane domain protein with both its termini located in the cytoplasm. Moreover, the conserved loop cysteines, which are not required for VKORC1 activity, are essential for VKORC1L1''s active site regeneration. Results from domain exchanges between VKORC1L1 and VKORC1 suggest that it is VKORC1L1''s overall structure that uniquely allows for active site regeneration by the conserved loop cysteines. Intermediate disulfide trapping results confirmed an intra-molecular electron transfer pathway for VKORC1L1''s active site reduction. Our results allow us to propose a concerted action of the four conserved cysteines of VKORC1L1 for active site regeneration; the second loop cysteine, Cys-58, attacks the active site disulfide, forming an intermediate disulfide with Cys-139; the first loop cysteine, Cys-50, attacks the intermediate disulfide resulting in active site reduction. The different membrane topologies and reaction mechanisms between VKORC1L1 and VKORC1 suggest that these two proteins might have different physiological functions.     

Preferential targeting of a signal recognition particle-dependent precursor to the Ssh1p translocon in yeast

Protein translocation across the endoplasmic reticulum membrane occurs via a "translocon" channel formed by the Sec61p complex. In yeast, two channels exist: the canonical Sec61p channel and a homolog called Ssh1p. Here, we used trapped translocation intermediates to demonstrate that a specific signal recognition particle-dependent substrate, Sec71p, is targeted exclusively to Ssh1p. Strikingly, we found that, in the absence of Ssh1p, precursor could be successfully redirected to canonical Sec61p, demonstrating that the normal targeting reaction must involve preferential sorting to Ssh1p. Our data therefore demonstrate that Ssh1p is the primary translocon for Sec71p and reveal a novel sorting mechanism at the level of the endoplasmic reticulum membrane enabling precursors to be directed to distinct translocons. Interestingly, the Ssh1p-dependent translocation of Sec71p was found to be dependent upon Sec63p, demonstrating a previously unappreciated functional interaction between Sec63p and the Ssh1p translocon.     

The role of ERp44 in maturation of serotonin transporter protein

In heterologous and endogenous expression systems, we studied the role of ERp44 and its complex partner endoplasmic reticulum (ER) oxidase 1-α (Ero1-Lα) in mechanisms regulating disulfide bond formation for serotonin transporter (SERT), an oligomeric glycoprotein. ERp44 is an ER lumenal chaperone protein that favors the maturation of disulfide-linked oligomeric proteins. ERp44 plays a critical role in the release of proteins from the ER via binding to Ero1-Lα. Mutation in the thioredoxin-like domain hampers the association of ERp44C29S with SERT, which has three Cys residues (Cys-200, Cys-209, and Cys-109) on the second external loop. We further explored the role of the protein chaperones through shRNA knockdown experiments for ERp44 and Ero1-Lα. Those efforts resulted in increased SERT localization to the plasma membrane but decreased serotonin (5-HT) uptake rates, indicating the importance of the ERp44 retention mechanism in the proper maturation of SERT proteins. These data were strongly supported with the data received from the N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin) labeling of SERT on ERp44 shRNA cells. MTSEA-biotin only interacts with the free Cys residues from the external phase of the plasma membrane. Interestingly, it appears that Cys-200 and Cys-209 of SERT in ERp44-silenced cells are accessible to labeling by MTSEA-biotin. However, in the control cells, these Cys residues are occupied and produced less labeling with MTSEA-biotin. Furthermore, ERp44 preferentially associated with SERT mutants (C200S, C209S, and C109A) when compared with wild type. These interactions with the chaperone may reflect the inability of Cys-200 and Cys-209 SERT mutants to form a disulfide bond and self-association as evidenced by immunoprecipitation assays. Based on these collective findings, we hypothesize that ERp44 together with Ero1-Lα plays an important role in disulfide formation of SERT, which may be a prerequisite step for the assembly of SERT molecules in oligomeric form.     

The TOR Complex 1 Is Distributed in Endosomes and in Retrograde Vesicles That Form from the Vacuole Membrane and Plays an Important Role in the Vacuole Import and Degradation Pathway

The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is induced when Saccharomyces cerevisiae are starved of glucose. However, when glucose is added to cells that have been starved for 3 days, FBPase is degraded in the vacuole. FBPase is first imported to Vid (vacuole import and degradation) vesicles, and these vesicles then merge with the endocytic pathway. In this report we show that two additional gluconeogenic enzymes, isocitrate lyase and phosphoenolpyruvate carboxykinase, were also degraded in the vacuole via the Vid pathway. These new cargo proteins and FBPase interacted with the TORC1 complex during glucose starvation. However, Tor1p was dissociated from FBPase after the addition of glucose. FBPase degradation was inhibited in cells overexpressing TOR1, suggesting that excessive Tor1p is inhibitory. Both Tco89p and Tor1p were found in endosomes coming from the plasma membrane as well as in retrograde vesicles forming from the vacuole membrane. When TORC1 was inactivated by rapamycin, FBPase degradation was inhibited. We suggest that TORC1 interacts with multiple cargo proteins destined for the Vid pathway and plays an important role in the degradation of FBPase in the vacuole.     

N-terminal Extension of the Cholera Toxin A1-chain Causes Rapid Degradation after Retrotranslocation from Endoplasmic Reticulum to Cytosol

Cholera toxin travels from the plasma membrane to the endoplasmic reticulum of host cells, where a portion of the toxin, the A1-chain, is unfolded and targeted to a protein-conducting channel for retrotranslocation to the cytosol. Unlike most retrotranslocation substrates, the A1-chain escapes degradation by the proteasome and refolds in the cytosol to induce disease. How this occurs remains poorly understood. Here, we show that an unstructured peptide appended to the N terminus of the A1-chain renders the toxin functionally inactive. Cleavage of the peptide extension prior to cell entry rescues toxin half-life and function. The loss of toxicity is explained by rapid degradation by the proteasome after retrotranslocation to the cytosol. Degradation of the mutant toxin does not follow the N-end rule but depends on the two Lys residues at positions 4 and 17 of the native A1-chain, consistent with polyubiquitination at these sites. Thus, retrotranslocation and refolding of the wild-type A1-chain must proceed in a way that protects these Lys residues from attack by E3 ligases.     

Sec23 Homolog Nel1 Is a Novel GTPase-activating Protein for Sar1 but Does Not Function as a Subunit of the Coat Protein Complex II (COPII) Coat

The coat protein complex II (COPII) generates transport carriers from the endoplasmic reticulum (ER) under the control of the small GTPase Sar1. Sec23 is well known as a structural component of the COPII coat and as a GTPase-activating protein (GAP) for Sar1. Here, we showed that Saccharomyces cerevisiae contains a novel Sec23 paralog, Nel1, which appears not to function as a subunit of the COPII coat. Nel1 does not associate with any of the COPII components, but it exhibits strong Sar1 GAP activity. We also demonstrated that the chromosomal deletion of NEL1 leads to a significant growth defect in the temperature-sensitive sar1D32G background, suggesting a possible functional link between these proteins. In contrast to Sec23, which is predominantly localized at ER exit sites on the ER membrane, a major proportion of Nel1 is localized throughout the cytosol. Our findings highlight a possible role of Nel1 as a novel GAP for Sar1.     

A Tyrosine-based Motif Localizes a Drosophila Vesicular Transporter to Synaptic Vesicles in Vivo

Vesicular neurotransmitter transporters must localize to synaptic vesicles (SVs) to allow regulated neurotransmitter release at the synapse. However, the signals required to localize vesicular proteins to SVs in vivo remain unclear. To address this question we have tested the effects of mutating proposed trafficking domains in Drosophila orthologs of the vesicular monoamine and glutamate transporters, DVMAT-A and DVGLUT. We show that a tyrosine-based motif (YXXY) is important both for DVMAT-A internalization from the cell surface in vitro, and localization to SVs in vivo. In contrast, DVGLUT deletion mutants that lack a putative C-terminal trafficking domain show more modest defects in both internalization in vitro and trafficking to SVs in vivo. Our data show for the first time that mutation of a specific trafficking motif can disrupt localization to SVs in vivo and suggest possible differences in the sorting of VMATs versus VGLUTs to SVs at the synapse.     

Glycoprotein Biosynthesis in a Eukaryote Lacking the Membrane Protein Rft1

Mature dolichol-linked oligosaccharides (mDLOs) needed for eukaryotic proteinN-glycosylation are synthesized by a multistep pathway in which the biosyntheticlipid intermediate Man₅GlcNAc₂-PP-dolichol (M5-DLO) flips from the cytoplasmicto the luminal face of the endoplasmic reticulum. The endoplasmic reticulum membrane protein Rft1 isintimately involved in mDLO biosynthesis. Yeast genetic analyses implicated Rft1 as the M5-DLOflippase, but because biochemical tests challenged this assignment, the function of Rft1 remainsobscure. To understand the role of Rft1, we sought to analyze mDLO biosynthesis invivo in the complete absence of the protein. Rft1 is essential for yeast viability, and noRft1-null organisms are currently available. Here, we exploited Trypanosoma brucei(Tb), an early diverging eukaryote whose Rft1 homologue functions in yeast. We report thatTbRft1-null procyclic trypanosomes grow nearly normally. They have normal steady-state levels ofmDLO and significant N-glycosylation, indicating robust M5-DLO flippase activity.Remarkably, the mutant cells have 30–100-fold greater steady-state levels of M5-DLO thanwild-type cells. All N-glycans in the TbRft1-null cells originate from mDLOindicating that the M5-DLO excess is not available for glycosylation. These results suggest thatrather than facilitating M5-DLO flipping, Rft1 facilitates conversion of M5-DLO to mDLO by anothermechanism, possibly by acting as an M5-DLO chaperone.     

Filling the Gap, Evolutionarily Conserved Omp85 in Plastids of Chromalveolates

Chromalveolates are a diverse group of protists that include many ecologically and medically relevant organisms such as diatoms and apicomplexan parasites. They possess plastids generally surrounded by four membranes, which evolved by engulfment of a red alga. Today, most plastid proteins must be imported, but many aspects of protein import into complex plastids are still cryptic. In particular, how proteins cross the third outermost membrane has remained unexplained. We identified a protein in the third outermost membrane of the diatom Phaeodactylum tricornutum with properties comparable to those of the Omp85 family. We demonstrate that the targeting route of P. tricornutum Omp85 parallels that of the translocation channel of the outer envelope membrane of chloroplasts, Toc75. In addition, the electrophysiological properties are similar to those of the Omp85 proteins involved in protein translocation. This supports the hypothesis that P. tricornutum Omp85 is involved in precursor protein translocation, which would close a gap in the fundamental understanding of the evolutionary origin and function of protein import in secondary plastids.     

Identification of Glycosyltransferase 8 Family Members as Xylosyltransferases Acting on O-Glucosylated Notch Epidermal Growth Factor Repeats

The epidermal growth factor repeats of the Notch receptor are extensively glycosylated with three different O-glycans. O-Fucosylation and elongation by the glycosyltransferase Fringe have been well studied and shown to be essential for proper Notch signaling. In contrast, biosynthesis of O-glucose and O-N-acetylglucosamine is less well understood. Recently, the isolation of the Drosophila mutant rumi has shown that absence of O-glucose impairs Notch function. O-Glucose is further extended by two contiguous α1,3-linked xylose residues. We have identified two enzymes of the human glycosyltransferase 8 family, now named GXYLT1 and GXYLT2 (glucoside xylosyltransferase), as UDP-d-xylose:β-d-glucoside α1,3-d-xylosyltransferases adding the first xylose. The enzymes are specific for β-glucose-terminating acceptors and UDP-xylose as donor substrate. Generation of the α1,3-linkage was confirmed by nuclear magnetic resonance. Activity on a natural acceptor could be shown by in vitro xylosylation of a Notch fragment expressed in a UDP-xylose-deficient cell line and in vivo by co-expression of the enzymes and the Notch fragment in insect cells followed by mass spectrometric analysis of peptide fragments.     

Evidence That the Yeast Desaturase Ole1p Exists as a Dimer in Vivo

Desaturase enzymes are composed of two classes, the structurally well characterized soluble class found predominantly in the plastids of higher plants and the more widely distributed but poorly structurally defined integral membrane class. Despite their distinct evolutionary origins, the two classes both require an iron cofactor and molecular oxygen for activity and are inhibited by azide and cyanide, suggesting strong mechanistic similarities. The fact that the soluble desaturase is active as a homodimer prompted us test the hypothesis that an archetypal integral membrane desaturase from Saccharomyces cerevisiae, the Δ⁹-acyl-Co-A desaturase Ole1p, also exhibits a dimeric organization. Ole1p was chosen because it is one of the best characterized integral membrane desaturase and because it retains activity when fused with epitope tags. FLAG-Ole1p was detected by Western blotting of immunoprecipitates in which anti-Myc antibodies were used for capture from yeast extracts co-expressing Ole1p-Myc and Ole1p-FLAG. Interaction was confirmed by two independent bimolecular complementation assays (i.e. the split ubiquitin system and the split luciferase system). Co-expression of active and inactive Ole1p subunits resulted in an ∼75% suppression of the accumulation of palmitoleic acid, demonstrating that the physiologically active form of Ole1p in vivo is the dimer in which both protomers must be functional.     

Usa1p Is Required for Optimal Function and Regulation of the Hrd1p Endoplasmic Reticulum-associated Degradation Ubiquitin Ligase

Usa1p is a recently discovered member of the HRD ubiquitin ligase complex. The HRD pathway is a conserved route of ubiquitin-dependent, endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous lumenal (ERAD-L) and membrane-anchored (ERAD-M) substrates. We have investigated Usa1p to understand its importance in HRD complex action. Usa1p was required for the optimal function of the Hrd1p E3 ubiquitin ligase; its loss caused deficient degradation of both membrane-associated and lumenal proteins. Furthermore, Usa1p functioned in regulation of Hrd1p by two mechanisms. First, Hrd1p self-degradation, which serves to limit the levels of uncomplexed E3, is absolutely dependent on Usa1p and the ubiquitin-like (Ubl) domain of Usa1p. We found that Usa1p allows Hrd1p degradation by promoting trans interactions between Hrd1p molecules. The Ubl domain of Usa1p was required specifically for Hrd1p self-ubiquitination but not for degradation of either ERAD-L or ERAD-M substrates. In addition, Usa1p was able to attenuate the activity-dependent toxicity of Hrd1p without compromising substrate degradation, indicating a separate role in ligase regulation that operates in parallel to stability control. Many of the described actions of Usa1p are distinct from those of Der1p, which is recruited to the HRD complex by Usa1p. Thus, this novel, conserved factor is broadly involved in the function and regulation of the HRD pathway of ERAD.     

Sec62 Protein Mediates Membrane Insertion and Orientation of Moderately Hydrophobic Signal Anchor Proteins in the Endoplasmic Reticulum (ER)

Nascent chains are known to be targeted to the endoplasmic reticulum membrane either by a signal recognition particle (SRP)-dependent co-translational or by an SRP-independent post-translational translocation route depending on signal sequences. Using a set of model and cellular proteins carrying an N-terminal signal anchor sequence of controlled hydrophobicity and yeast mutant strains defective in SRP or Sec62 function, the hydrophobicity-dependent targeting efficiency and targeting pathway preference were systematically evaluated. Our results suggest that an SRP-dependent co-translational and an SRP-independent post-translational translocation are not mutually exclusive for signal anchor proteins and that moderately hydrophobic ones require both SRP and Sec62 for proper targeting and translocation to the endoplasmic reticulum. Further, defect in Sec62 selectively reduced signal sequences inserted in an N_in-C_out (type II) membrane topology, implying an undiscovered role of Sec62 in regulating the orientation of the signal sequence in an early stage of translocation.     

Regulation of Phagocytosis in Macrophages by Neuraminidase 1

The differentiation of monocytes into macrophages and dendritic cells is accompanied by induction of cell-surface neuraminidase 1 (Neu1) and cathepsin A (CathA), the latter forming a complex with and activating Neu1. To clarify the biological importance of this phenomenon we have developed the gene-targeted mouse models of a CathA deficiency (CathA^S190A) and a double CathA/Neu1 deficiency (CathA^S190A-Neo). Macrophages of CathA^S190A-Neo mice and their immature dendritic cells showed a significantly reduced capacity to engulf Gram-positive and Gram-negative bacteria and positively and negatively charged polymer beads as well as IgG-opsonized beads and erythrocytes. Properties of the cells derived from CathA^S190A mice were indistinguishable from those of wild-type controls, suggesting that the absence of Neu1, which results in the increased sialylation of the cell surface proteins, probably affects multiple receptors for phagocytosis. Indeed, treatment of the cells with purified mouse Neu1 reduced surface sialylation and restored phagocytosis. Because Neu1-deficient cells showed reduced internalization of IgG-opsonized sheep erythrocytes whereas binding of the erythrocytes to the cells at 4 °C persisted, we speculate that the absence of Neu1 in particular affected transduction of signals from the Fc receptors for immunoglobulin G (FcγR). Indeed the macrophages from the Neu1-deficient mice showed increased sialylation and impaired phosphorylation of FcγR as well as markedly reduced phosphorylation of Syk kinase in response to treatment with IgG-opsonized beads. Altogether our data suggest that the cell surface Neu1 activates the phagocytosis in macrophages and dendritic cells through desialylation of surface receptors, thus, contributing to their functional integrity.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.