Identification of a Novel Protein Binding Motif within the T-synthase for the Molecular Chaperone Cosmc

Prior studies suggested that the core 1 β3-galactosyltransferase (T-synthase) is a specific client of the endoplasmic reticulum chaperone Cosmc, whose function is required for T-synthase folding, activity, and consequent synthesis of normal O-glycans in all vertebrate cells. To explore whether the T-synthase encodes a specific recognition motif for Cosmc, we used deletion mutagenesis to identify a cryptic linear and relatively hydrophobic peptide in the N-terminal stem region of the T-synthase that is essential for binding to Cosmc (Cosmc binding region within T-synthase, or CBRT). Using this sequence information, we synthesized a peptide containing CBRT and found that it directly interacts with Cosmc and also inhibits Cosmc-assisted in vitro refolding of denatured T-synthase. Moreover, engineered T-synthase carrying mutations within CBRT exhibited diminished binding to Cosmc that resulted in the formation of inactive T-synthase. To confirm the general recognition of CBRT by Cosmc, we performed a domain swap experiment in which we inserted the stem region of the T-synthase into the human β4GalT1 and found that the CBRT element can confer Cosmc binding onto the β4GalT1 chimera. Thus, CBRT is a unique recognition motif for Cosmc to promote its regulation and formation of active T-synthase and represents the first sequence-specific chaperone recognition system in the ER/Golgi required for normal protein O-glycosylation.     

Post-translational modifications of the gamma-subunit affect intracellular trafficking and complex assembly of GlcNAc-1-phosphotransferase

GlcNAc-1-phosphotransferase plays a key role in the generation of mannose 6-phosphate, a recognition marker essential for efficient transport of lysosomal hydrolases to lysosomes. The enzyme complex is composed of six subunits (α(2)β(2)γ(2)). The α- and β-subunits are catalytically active, whereas the function of the γ-subunit is still unclear. We have investigated structural properties, localization, and intracellular transport of the human and mouse γ-subunits and the molecular requirements for the assembly of the phosphotransferase complex. The results showed that endogenous and overexpressed γ-subunits were localized in the cis-Golgi apparatus. Secreted forms of γ-subunits were detectable in media of cultured cells as well as in human serum. The γ-subunit contains two in vivo used N-glycosylation sites at positions 88 and 115, equipped with high mannose-type oligosaccharides. (35)S pulse-chase experiments and size exclusion chromatography revealed that the majority of non-glycosylated γ-subunit mutants were integrated in high molecular mass complexes, failed to exit the endoplasmic reticulum (ER), and were rapidly degraded. The substitution of cysteine 245 involved in dimerization of γ-subunits impaired neither ER exit nor trafficking through the secretory pathway. Monomeric γ-subunits failed, however, to associate with other GlcNAc-1-phosphotransferase subunits. The data provide evidence that assembly of the GlcNAc-1-phosphotransferase complex takes place in the ER and requires dimerization of the γ-subunits.     

Identification of an Htm1 (EDEM)-dependent,Mns1-independent Endoplasmic Reticulum-associated Degradation (ERAD) Pathway in Saccharomyces cerevisiae: APPLICATION OF A NOVEL ASSAY FOR GLYCOPROTEIN ERAD*

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a quality control system for newly synthesized proteins in the ER; nonfunctional proteins, which fail to form their correct folding state, are then degraded. The cytoplasmic peptide:N-glycanase is a deglycosylating enzyme that is involved in the ERAD and releases N-glycans from misfolded glycoproteins/glycopeptides. We have previously identified a mutant plant toxin protein, RTA (ricin A-chain nontoxic mutant), as the first in vivo Png1 (the cytoplasmic peptide:N-glycanase in Saccharomyces cerevisiae)-dependent ERAD substrate. Here, we report a new genetic device to assay the Png1-dependent ERAD pathway using the new model protein designated RTL (RTA-transmembrane-Leu2). Our extensive studies using different yeast mutants identified various factors involved in RTL degradation. The degradation of RTA/RTL was independent of functional Sec61 but was dependent on Der1. Interestingly, ER-mannosidase Mns1 was not involved in RTA degradation, but it was dependent on Htm1 (ERAD-related α-mannosidase in yeast) and Yos9 (a putative degradation lectin), indicating that mannose trimming by Mns1 is not essential for efficient ERAD of RTA/RTL. The newly established RTL assay will allow us to gain further insight into the mechanisms involved in the Png1-dependent ERAD-L pathway.     

Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ～20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.     

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis

Nucleotide sugar transporters of the Golgi apparatus play an essential role in the glycosylation of proteins, lipids, and proteoglycans. Down-regulation of expression of the transporters for CMP-sialic acid, GDP-fucose, or both unexpectedly resulted in accumulation of glycoconjugates in the Golgi apparatus rather than in the plasma membrane. Pulse-chase experiments with radiolabeled sugars and amino acids showed decreased synthesis and secretion of both nonglycoproteins and glycoproteins. Further studies revealed that the above silencing induced endoplasmic reticulum stress and inhibited protein translation initiation. Together these results suggest that global inhibition of Golgi apparatus glycosylation may lead to important secondary metabolic changes, unrelated to glycosylation.     

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.     

Endoplasmic reticulum-associated degradation (ERAD) and free oligosaccharide generation in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, proteins with misfolded lumenal, membrane, and cytoplasmic domains are cleared from the endoplasmic reticulum (ER) by ER-associated degradation (ERAD)-L, -M, and -C, respectively. ERAD-L is N-glycan-dependent and is characterized by ER mannosidase (Mns1p) and ER mannosidase-like protein (Mnl1p), which generate Man(7)GlcNAc(2) (d1) N-glycans with non-reducing α1,6-mannosyl residues. Glycoproteins bearing this motif bind Yos9p and are dislocated into the cytoplasm and then deglycosylated by peptide N-glycanase (Png1p) to yield free oligosaccharides (fOS). Here, we examined yeast fOS metabolism as a function of cell growth in order to obtain quantitative and mechanistic insights into ERAD. We demonstrate that both Png1p-dependent generation of Man(7-10)GlcNAc(2) fOS and vacuolar α-mannosidase (Ams1p)-dependent fOS demannosylation to yield Man(1)GlcNAc(2) are strikingly up-regulated during post-diauxic growth which occurs when the culture medium is depleted of glucose. Gene deletions in the ams1Δ background revealed that, as anticipated, Mns1p and Mnl1p are required for efficient generation of the Man(7)GlcNAc(2) (d1) fOS, but for the first time, we demonstrate that small amounts of this fOS are generated in an Mnl1p-independent, Mns1p-dependent pathway and that a Man(8)GlcNAc(2) fOS that is known to bind Yos9p is generated in an Mnl1p-dependent, Mns1p-independent manner. This latter observation adds mechanistic insight into a recently described Mnl1p-dependent, Mns1p-independent ERAD pathway. Finally, we show that 50% of fOS generation is independent of ERAD-L, and because our data indicate that ERAD-M and ERAD-C contribute little to fOS levels, other important processes underlie fOS generation in S. cerevisiae.     

Phosphorylation of Serine Palmitoyltransferase Long Chain-1 (SPTLC1) on Tyrosine 164 Inhibits Its Activity and Promotes Cell Survival

In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr¹⁶⁴ by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr¹⁶⁴ to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.     

SEL1L protein critically determines the stability of the HRD1-SEL1L endoplasmic reticulum-associated degradation (ERAD) complex to optimize the degradation kinetics of ERAD substrates

The mammalian HRD1-SEL1L complex provides a scaffold for endoplasmic reticulum (ER)-associated degradation (ERAD), thereby connecting luminal substrates for ubiquitination at the cytoplasmic surface after their retrotranslocation through the endoplasmic reticulum membrane. In this study the stability of the mammalian HRD1-SEL1L complex was assessed by performing siRNA-mediated knockdown of each of its components. Although endogenous SEL1L is a long-lived protein, the half-life of SEL1L was greatly reduced when HRD1 is silenced. Conversely, transiently expressed SEL1L was rapidly degraded but was stabilized when HRD1 was coexpressed. This was in contrast to the yeast Hrd1p-Hrd3p, where Hrd1p is destabilized by the depletion of Hrd3p, the SEL1L homologue. Endogenous HRD1-SEL1L formed a large ERAD complex (Complex I) associating with numerous ERAD components including ERAD lectin OS-9, membrane-spanning Derlin-1/2, VIMP, and Herp, whereas transiently expressed HRD1-SEL1L formed a smaller complex (Complex II) that was associated with OS-9 but not with Derlin-1/2, VIMP, or Herp. Despite its lack of stable association with the latter components, Complex II supported the retrotranslocation and degradation of model ERAD substrates α1-antitrypsin null Hong-Kong (NHK) and its variant NHK-QQQ lacking the N-glycosylation sites. NHK-QQQ was rapidly degraded when SEL1L was transiently expressed, whereas the simultaneous transfection of HRD1 diminished that effect. SEL1L unassociated with HRD1 was degraded by the ubiquitin-proteasome pathway, which suggests the involvement of a ubiquitin-ligase other than HRD1 in the rapid degradation of both SEL1L and NHK-QQQ. These results indicate that the regulation of the stability and assembly of the HRD1-SEL1L complex is critical to optimize the degradation kinetics of ERAD substrates.     

Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoan Spo7p ortholog and functions in the lipin activation pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.     

In vivo crystallization of human IgG in the endoplasmic reticulum of engineered Chinese hamster ovary (CHO) cells

Protein synthesis and secretion are essential to cellular life. Although secretory activities may vary in different cell types, what determines the maximum secretory capacity is inherently difficult to study. Increasing protein synthesis until reaching the limit of secretory capacity is one strategy to address this key issue. Under highly optimized growth conditions, recombinant CHO cells engineered to produce a model human IgG clone started housing rod-shaped crystals in the endoplasmic reticulum (ER) lumen. The intra-ER crystal growth was accompanied by cell enlargement and multinucleation and continued until crystals outgrew cell size to breach membrane integrity. The intra-ER crystals were composed of correctly folded, endoglycosidase H-sensitive IgG. Crystallizing propensity was due to the intrinsic physicochemical properties of the model IgG, and the crystallization was reproduced in vitro by exposing a high concentration of IgG to a near neutral pH. The striking cellular phenotype implicated the efficiency of IgG protein synthesis and oxidative folding exceeded the capacity of ER export machinery. As a result, export-ready IgG accumulated progressively in the ER lumen until a threshold concentration was reached to nucleate crystals. Using an in vivo system that reports accumulation of correctly folded IgG, we showed that the ER-to-Golgi transport steps became rate-limiting in cells with high secretory activity.     

Structural Characterization of Carbohydrate Binding by LMAN1 Protein Provides New Insight into the Endoplasmic Reticulum Export of Factors V (FV) and VIII (FVIII)

LMAN1 (ERGIC-53) is a key mammalian cargo receptor responsible for the export of a subset of glycoproteins from the endoplasmic reticulum. Together with its soluble coreceptor MCFD2, LMAN1 transports coagulation factors V (FV) and VIII (FVIII). Mutations in LMAN1 or MCFD2 cause the genetic bleeding disorder combined deficiency of FV and FVIII (F5F8D). The LMAN1 carbohydrate recognition domain (CRD) binds to both glycoprotein cargo and MCFD2 in a Ca²⁺-dependent manner. To understand the biochemical basis and regulation of LMAN1 binding to glycoprotein cargo, we solved crystal structures of the LMAN1-CRD bound to Man-α-1,2-Man, the terminal carbohydrate moiety of high mannose glycans. Our structural data, combined with mutagenesis and in vitro binding assays, define the central mannose-binding site on LMAN1 and pinpoint histidine 178 and glycines 251/252 as critical residues for FV/FVIII binding. We also show that mannobiose binding is relatively independent of pH in the range relevant for endoplasmic reticulum-to-Golgi traffic, but is sensitive to lowered Ca²⁺ concentrations. The distinct LMAN1/MCFD2 interaction is maintained at these lowered Ca²⁺ concentrations. Our results suggest that compartmental changes in Ca²⁺ concentration regulate glycoprotein cargo binding and release from the LMAN1·MCFD2 complex in the early secretory pathway.     

A Small Molecule Inhibitor of Endoplasmic Reticulum Oxidation 1 (ERO1) with Selectively Reversible Thiol Reactivity

Endoplasmic reticulum oxidation 1 (ERO1) is a conserved eukaryotic flavin adenine nucleotide-containing enzyme that promotes disulfide bond formation by accepting electrons from reduced protein disulfide isomerase (PDI) and passing them on to molecular oxygen. Although disulfide bond formation is an essential process, recent experiments suggest a surprisingly broad tolerance to genetic manipulations that attenuate the rate of disulfide bond formation and that a hyperoxidizing ER may place stressed cells at a disadvantage. In this study, we report on the development of a high throughput in vitro assay for mammalian ERO1α activity and its application to identify small molecule inhibitors. The inhibitor EN460 (IC₅₀, 1.9 μm) interacts selectively with the reduced, active form of ERO1α and prevents its reoxidation. Despite rapid and promiscuous reactivity with thiolates, EN460 exhibits selectivity for ERO1. This selectivity is explained by the rapid reversibility of the reaction of EN460 with unstructured thiols, in contrast to the formation of a stable bond with ERO1α followed by displacement of bound flavin adenine dinucleotide from the active site of the enzyme. Modest concentrations of EN460 and a functionally related inhibitor, QM295, promote signaling in the unfolded protein response and precondition cells against severe ER stress. Together, these observations point to the feasibility of targeting the enzymatic activity of ERO1α with small molecule inhibitors.     

The Dsl1 tethering complex actively participates in soluble NSF (N-ethylmaleimide-sensitive factor) attachment protein receptor (SNARE) complex assembly at the endoplasmic reticulum in Saccharomyces cerevisiae

Intracellular transport is largely dependent on vesicles that bud off from one compartment and fuse with the target compartment. The first contact of an incoming vesicle with the target membrane is mediated by tethering factors. The tethering factor responsible for recruiting Golgi-derived vesicles to the ER is the Dsl1 tethering complex, which is comprised of the essential proteins Dsl1p, Dsl3p, and Tip20p. We investigated the role of the Tip20p subunit at the ER by analyzing two mutants, tip20-5 and tip20-8. Both mutants contained multiple mutations that were scattered throughout the TIP20 sequence. Individual mutations could not reproduce the temperature-sensitive phenotype of tip20-5 and tip20-8, indicating that the overall structure of Tip20p might be altered in the mutants. Using molecular dynamics simulations comparing Tip20p and Tip20-8p revealed that some regions, particularly the N-terminal domain and parts of the stalk region, were more flexible in the mutant protein, consistent with its increased susceptibility to proteolysis. Both Tip20-5p and Tip20-8p mutants prevented proper ER trans-SNARE complex assembly in vitro. Moreover, Tip20p mutant proteins disturbed the interaction between Dsl1p and the coatomer coat complex, indicating that the Dsl1p-coatomer interaction could be stabilized or regulated by Tip20p. We provide evidence for a direct role of the Dsl1 complex, in particular Tip20p, in the formation and stabilization of ER SNARE complexes.     

Isoform- and Species-specific Control of Inositol 1,4,5-Trisphosphate (IP3) Receptors by Reactive Oxygen Species

Reactive oxygen species (ROS) stimulate cytoplasmic [Ca²⁺] ([Ca²⁺]_c) signaling, but the exact role of the IP₃ receptors (IP₃R) in this process remains unclear. IP₃Rs serve as a potential target of ROS produced by both ER and mitochondrial enzymes, which might locally expose IP₃Rs at the ER-mitochondrial associations. Also, IP₃Rs contain multiple reactive thiols, common molecular targets of ROS. Therefore, we have examined the effect of superoxide anion (O₂^⨪) on IP₃R-mediated Ca²⁺ signaling. In human HepG2, rat RBL-2H3, and chicken DT40 cells, we observed [Ca²⁺]_c spikes and frequency-modulated oscillations evoked by a O₂^⨪ donor, xanthine (X) + xanthine oxidase (XO), dose-dependently. The [Ca²⁺]_c signal was mediated by ER Ca²⁺ mobilization. X+XO added to permeabilized cells promoted the [Ca²⁺]_c rise evoked by submaximal doses of IP₃, indicating that O₂^⨪ directly sensitizes IP₃R-mediated Ca²⁺ release. In response to X+XO, DT40 cells lacking two of three IP₃R isoforms (DKO) expressing either type 1 (DKO1) or type 2 IP₃Rs (DKO2) showed a [Ca²⁺]_c signal, whereas DKO expressing type 3 IP₃R (DKO3) did not. By contrast, IgM that stimulates IP₃ formation, elicited a [Ca²⁺]_c signal in every DKO. X+XO also facilitated the Ca²⁺ release evoked by submaximal IP₃ in permeabilized DKO1 and DKO2 but was ineffective in DKO3 or in DT40 lacking every IP₃R (TKO). However, X+XO could also facilitate the effect of suboptimal IP₃ in TKO transfected with rat IP₃R3. Although in silico studies failed to identify a thiol missing in the chicken IP₃R3, an X+XO-induced redox change was documented only in the rat IP₃R3. Thus, ROS seem to specifically sensitize IP₃Rs through a thiol group(s) within the IP₃R, which is probably inaccessible in the chicken IP₃R3.     

Mannose trimming is required for delivery of a glycoprotein from EDEM1 to XTP3-B and to late endoplasmic reticulum-associated degradation steps

Although the trimming of α1,2-mannose residues from precursor N-linked oligosaccharides is an essential step in the delivery of misfolded glycoproteins to endoplasmic reticulum (ER)-associated degradation (ERAD), the exact role of this trimming is unclear. EDEM1 was initially suggested to bind N-glycans after mannose trimming, a role presently ascribed to the lectins OS9 and XTP3-B, because of their in vitro affinities for trimmed oligosaccharides. We have shown before that ER mannosidase I (ERManI) is required for the trimming and concentrates together with the ERAD substrate and ERAD machinery in the pericentriolar ER-derived quality control compartment (ERQC). Inhibition of mannose trimming prevents substrate accumulation in the ERQC. Here, we show that the mannosidase inhibitor kifunensine or ERManI knockdown do not affect binding of an ERAD substrate glycoprotein to EDEM1. In contrast, substrate association with XTP3-B and with the E3 ubiquitin ligases HRD1 and SCF(Fbs2) was inhibited. Consistently, whereas the ERAD substrate partially colocalized upon proteasomal inhibition with EDEM1, HRD1, and Fbs2 at the ERQC, colocalization was repressed by mannosidase inhibition in the case of the E3 ligases but not for EDEM1. Interestingly, association and colocalization of the substrate with Derlin-1 was independent of mannose trimming. The HRD1 adaptor protein SEL1L had been suggested to play a role in N-glycan-dependent substrate delivery to OS9 and XTP3-B. However, substrate association with XTP3-B was still dependent on mannose trimming upon SEL1L knockdown. Our results suggest that mannose trimming enables delivery of a substrate glycoprotein from EDEM1 to late ERAD steps through association with XTP3-B.     

Proteomic assessment shows that many endoplasmic reticulum (ER)-resident proteins are targeted by N(epsilon)-lysine acetylation in the lumen of the organelle and predicts broad biological impact

In addition to the nucleus, cytosol, and mitochondrial lumen, N(ε)-lysine acetylation also occurs in the lumen of the endoplasmic reticulum (ER). However, the impact of such an event on ER functions is still unknown. Here, we analyzed the "ER acetyl-lysine proteome" by nano-LC-MS/MS and discovered that a large number of ER-resident and -transiting proteins undergo N(ε)-lysine acetylation in the lumen of the organelle. The list of ER-resident proteins includes chaperones and enzymes involved with post-translational modification and folding. Grouping of all acetylated proteins into major functional categories suggests that the ER-based acetylation machinery regulates very diverse biological events. As such, it is predicted to play a fundamental role in human physiology as well as human pathology.     

Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.     

Topology of 1-acyl-sn-glycerol-3-phosphate acyltransferases SLC1 and ALE1 and related membrane-bound O-acyltransferases (MBOATs) of Saccharomyces cerevisiae

In yeast, phosphatidic acid, the biosynthetic precursor for all glycerophospholipids and triacylglycerols, is made de novo by the 1-acyl-sn-glycerol-3-phosphate acyltransferases Ale1p and Slc1p. Ale1p belongs to the membrane-bound O-acyltransferase (MBOAT) family, which contains many enzymes acylating lipids but also others that acylate secretory proteins residing in the lumen of the ER. A histidine present in a very short loop between two predicted transmembrane domains is the only residue that is conserved throughout the MBOAT gene family. The yeast MBOAT proteins of known function comprise Ale1p, the ergosterol acyltransferases Are1p and Are2p, and Gup1p, the last of which acylates lysophosphatidylinositol moieties of GPI anchors on ER lumenal GPI proteins. C-terminal topology reporters added to truncated versions of Gup1p yield a topology predicting a lumenal location of its uniquely conserved histidine 447 residue. The same approach shows that Ale1p and Are2p also have the uniquely conserved histidine residing in the ER lumen. Because these data raised the possibility that phosphatidic acid could be made in the lumen of the ER, we further investigated the topology of the second yeast 1-acyl-sn-glycerol-3-phosphate acyltransferase, Slc1p. The location of C-terminal topology reporters, microsomal assays probing the protease sensitivity of inserted tags, and the accessibility of natural or artificially inserted cysteines to membrane-impermeant alkylating agents all indicate that the most conserved motif containing the presumed active site histidine of Slc1p is oriented toward the ER lumen, whereas other conserved motifs are cytosolic. The implications of these findings are discussed.