O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.     

Eukaryotic Oligosaccharyltransferase Generates Free Oligosaccharides during N-Glycosylation

Asparagine (N)-linked glycosylation regulates numerous cellular activities, such as glycoprotein quality control, intracellular trafficking, and cell-cell communications. In eukaryotes, the glycosylation reaction is catalyzed by oligosaccharyltransferase (OST), a multimembrane protein complex that is localized in the endoplasmic reticulum (ER). During N-glycosylation in the ER, the protein-unbound form of oligosaccharides (free oligosaccharides; fOSs), which is structurally related to N-glycan, is released into the ER lumen. However, the enzyme responsible for this process remains unidentified. Here, we demonstrate that eukaryotic OST generates fOSs. Biochemical and genetic analyses using mutant strains of Saccharomyces cerevisiae revealed that the generation of fOSs is tightly correlated with the N-glycosylation activity of OST. Furthermore, we present evidence that the purified OST complex can generate fOSs by hydrolyzing dolichol-linked oligosaccharide, the glycan donor substrate for N-glycosylation. The heterologous expression of a single subunit of OST from the protozoan Leishmania major in S. cerevisiae demonstrated that this enzyme functions both in N-glycosylation and generation of fOSs. This study provides insight into the mechanism of PNGase-independent formation of fOSs.     

Cotranslational Targeting and Posttranslational Translocation can Cooperate in Spc3 Topogenesis

Secretory and membrane proteins follow either the signal recognition particle (SRP)-dependent cotranslational translocation pathway or the SRP-independent Sec62/Sec63-dependent posttranslational pathway for their translocation across the endoplasmic reticulum (ER). However, increasing evidence suggests that most proteins are cotranslationally targeted to the ER, suggesting mixed mechanisms. It remains unclear how these two pathways cooperate. Previous studies have shown that Spc3, a signal-anchored protein, requires SRP and Sec62 for its biogenesis. This study investigated the targeting and topogenesis of Spc3 and the step at which SRP and Sec62 act using in vivo and in vitro translocation assays and co-immunoprecipitation. Our data suggest that Spc3 reaches its final topology in two steps: it enters the ER lumen head-first and then inverts its orientation. The first step is partially dependent on SRP, although independent of the Sec62/Sec63 complex. The second step is mediated by the Sec62/Sec63 complex. These data suggest that SRP and Sec62 act on a distinct step in the topogenesis of Spc3.     

Botrytis cinerea Protein O-Mannosyltransferases Play Critical Roles in Morphogenesis,Growth, and Virulence

Protein O-glycosylation is crucial in determining the structure and function of numerous secreted and membrane-bound proteins. In fungi, this process begins with the addition of a mannose residue by protein O-mannosyltransferases (PMTs) in the lumen side of the ER membrane. We have generated mutants of the three Botrytis cinerea pmt genes to study their role in the virulence of this wide-range plant pathogen. B. cinerea PMTs, especially PMT2, are critical for the stability of the cell wall and are necessary for sporulation and for the generation of the extracellular matrix. PMTs are also individually required for full virulence in a variety of hosts, with a special role in the penetration of intact plant leaves. The most significant case is that of grapevine leaves, whose penetration requires the three functional PMTs. Furthermore, PMT2 also contributes significantly to fungal adherence on grapevine and tobacco leaves. Analysis of extracellular and membrane proteins showed significant changes in the pattern of protein secretion and glycosylation by the pmt mutants, and allowed the identification of new protein substrates putatively glycosylated by specific PMTs. Since plants do no possess these enzymes, PMTs constitute a promising target in the development of novel control strategies against B. cinerea.     

Proper insertion and topogenesis of membrane proteins in the ER depend on Sec63

BackgroundIn eukaryotic cells, biogenesis of proteins destined to the secretory pathway begins from the cytosol. Nascent chains are either co-translationally or post-translationally targeted to the endoplasmic reticulum (ER) and translocated across the membrane through the Sec61 complex. For the post-translational translocation, the Sec62/Sec63 complex is additionally required. Sec63, however, is also shown to mediate co-translational translocation of a subset of proteins, the types and characteristics of proteins that Sec63 mediates in translocation still await to be defined.MethodsTo overview the types of proteins that require Sec63 for the ER translocation, we prepared Sec63 mutant lacking the first 39 residues (Sec63_ΔN39) in yeast and assessed initial translocation efficiencies of diverse types of precursors in the sec63_ΔN39 strain by a 5 min metabolic labeling. By employing Blue-Native gel electrophoresis (BN-PAGE), stability of the SEC complex (Sec61 plus Sec62/Sec63 complexes) isolated from cells carrying the Sec63_ΔN39 mutant was examined.ResultsAmong the various translocation precursors tested, we found that proper sorting of single- and double-pass membrane proteins was severely impaired in addition to post-translational translocation precursor in the sec63_ΔN39 mutant strain. Stability of the SEC complex was compromised upon deletion of the N-terminal 39 residues.ConclusionsThe N-terminus of Sec63 is important for stability of the SEC complex and Sec63 is required for proper sorting of membrane proteins in vivo.General significanceSec63 is essential on insertion of membrane proteins.     

Polypeptide N-acetylgalactosaminyltransferase 18 non-catalytically regulates the ER homeostasis and O-glycosylation

Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner.     

GTDC2 modifies O-mannosylated α-dystroglycan in the endoplasmic reticulum to generate N-acetyl glucosamine epitopes reactive with CTD110.6 antibody

Hypoglycosylation is a common characteristic of dystroglycanopathy, which is a group of congenital muscular dystrophies. More than ten genes have been implicated in α-dystroglycanopathies that are associated with the defect in the O-mannosylation pathway. One such gene is GTDC2, which was recently reported to encode O-mannose β-1,4-N-acetylglucosaminyltransferase. Here we show that GTDC2 generates CTD110.6 antibody-reactive N-acetylglucosamine (GlcNAc) epitopes on the O-mannosylated α-dystroglycan (α-DG). Using the antibody, we show that mutations of GTDC2 identified in Walker–Warburg syndrome and alanine-substitution of conserved residues between GTDC2 and EGF domain O-GlcNAc transferase resulted in decreased glycosylation. Moreover, GTDC2-modified GlcNAc epitopes are localized in the endoplasmic reticulum (ER). These data suggested that GTDC2 is a novel glycosyltransferase catalyzing GlcNAcylation of O-mannosylated α-DG in the ER. CTD110.6 antibody may be useful to detect a specific form of GlcNAcylated O-mannose and to analyze defective O-glycosylation in α-dystroglycanopathies.     

Regulation of N-glycosylation and secretion of Isthmin-1 by its C-mannosylation

BackgroundC-mannosylation is a type of protein glycosylation. Human Isthmin-1 (ISM1) is a 52-kDa secreted protein with a thrombospondin type 1 repeat (TSR) domain, containing two consensus C-mannosylation sequences at Trp²²³ and Trp²²⁶. In this study, we sought to examine the role of C-mannosylation in the secretion of ISM1.MethodsWe established and cultured an ISM1-overexpressing HT1080 cell line and purified recombinant ISM1 for analysis from the conditioned medium by LC-MS/MS. Subcellular localization of ISM1 was observed by confocal fluorescence microscopy.ResultsWe found that ISM1 is C-mannosylated at Trp²²³ and Trp²²⁶ in the TSR domain. To determine the functions of the C-mannosylation of ISM1, we established a C-mannosylation-defective mutant ISM1-overexpressing HT1080 cell line and measured its secretion of ISM1. The secretion of ISM1 decreased significantly in this mutant ISM1-overexpressing line compared with wild-type cells. Furthermore, ISM1 was N-glycosylated only in these C-mannosylation-defective cells.ConclusionsISM1 is C-mannosylated in its TSR domain, and the status of the C-mannosylation of ISM1 affects its N-glycosylation.General significanceThe C-mannosylation of ISM1 regulates its N-glycosylation status.     

O-glycosylation of the non-canonical T-cadherin from rabbit skeletal muscle by single mannose residues

O-mannosylation is a vital protein modification. In humans, defective O-mannosylation of α-dystroglycan results in severe congenital muscular dystrophies. However, other proteins bearing this modification in vivo are still largely unknown. Here, we describe a highly reliable method combining glycosidase treatment with LC–MS analyses to identify mammalian O-mannosylated proteins from tissue sources. Our workflow identified T-cadherin (H-cadherin, CDH13) as a novel O-mannosylated protein. In contrast to known O-mannosylated proteins, single mannose residues (Man-α-Ser/Thr) are attached to this cell adhesion molecule. Conserved O-glycosylation sites in T-, E- and N-cadherins from different species, point to a general role of O-mannosyl glycans for cadherin function.     

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34⁺ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61α), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.The production of red blood cells involves the terminal differentiation of hematopoietic stem cells in the bone marrow followed by release into the peripheral blood (1, 2). Red blood cells remain in circulation for ∼120 days and require the prior production of abundant red cell-specific proteins including hemoglobin, cytoskeletal proteins, and membrane glycoproteins such as anion exchanger 1 (AE1)³ and glycophorin A (GPA). During differentiation, erythroid progenitor cells undergo extensive remodeling of their cytoskeleton and loss of nuclei and other organelles like the endoplasmic reticulum (ER). AE1 and GPA are known to be synthesized late in differentiation when these key cellular components are lost (3). The efficient biosynthesis of these red cell membrane glycoproteins, however, is expected to require robust ER assembly machinery involving protein translocation, N-glycosylation, and protein folding chaperones.The proper folding of membrane glycoproteins engages the quality control function of cytosolic and ER chaperone proteins (4, 5). Newly synthesized proteins undergo cycles of binding and release with chaperones, minimizing aggregation and facilitating folding. Chaperones also play a role in the retention and degradation of misfolded proteins and in apoptosis (6-8). The membrane-bound ER chaperone calnexin (CNX) and its luminal paralog calreticulin (CRT) interact with folding intermediates via their lectin and protein binding domains, thereby preventing aggregation (9). A wide variety of glycoprotein substrates have been identified, with some binding to one or both chaperones, and both have been shown to be vital in the prevention of aggregation and proper maturation of membrane glycoproteins (9, 10). Disruption of interactions with CNX and CRT can allow misfolded membrane glycoproteins to escape the ER and traffic to the plasma membrane (9).In the present study, we examined the integrity of the ER protein translocation, N-glycosylation, and quality control machinery during the differentiation of human CD34⁺ erythroid cells in culture. We found that specific components of the protein quality control system were completely lost (CNX and ERp57) or diminished (Hsc70 and Hsp70) before the production of the major glycoproteins, AE1 and GPA, was completed. Components of the protein translocation (Sec61α) and N-glycosylation machinery (OST48) were, however, maintained. Chaperones that play other roles in erythrocyte maturation and survival (CRT, BiP, and Hsp90) were also retained (11). AE1 was found to traffic efficiently to the plasma membrane in mouse embryonic fibroblasts completely lacking the ER chaperone CNX or CRT. Furthermore, disruption of CNX/CRT-glycoprotein interactions in human K562 cells did not affect the cell-surface expression of GPA or AE1. These results demonstrate that CNX and ERp57 are not required for the efficient synthesis and folding of red cell membrane glycoproteins during terminal erythropoiesis. The lack of engagement with the quality control and disulfide folding machinery may allow the more rapid production of red cell glycoproteins late in differentiation, sacrificing quality for quantity.     

Improvement of N-glycan site occupancy of therapeutic glycoproteins produced in Pichia pastoris

Yeast is capable of performing posttranslational modifications, such as N- or O-glycosylation. It has been demonstrated that N-glycans play critical biological roles in therapeutic glycoproteins by modulating pharmacokinetics and pharmacodynamics. However, N-glycan sites on recombinant glycoproteins produced in yeast can be underglycosylated, and hence, not completely occupied. Genomic homology analysis indicates that the Pichia pastoris oligosaccharyltransferase (OST) complex consists of multiple subunits, including OST1, OST2, OST3, OST4, OST5, OST6, STT3, SWP1, and WBP1. Monoclonal antibodies produced in P. pastoris show that N-glycan site occupancy ranges from 75–85 % and is affected mainly by the OST function, and in part, by process conditions. In this study, we demonstrate that N-glycan site occupancy of antibodies can be improved to greater than 99 %, comparable to that of antibodies produced in mammalian cells (CHO), by overexpressing Leishmania major STT3D (LmSTT3D) under the control of an inducible alcohol oxidase 1 (AOX1) promoter. N-glycan site occupancy of non-antibody glycoproteins such as recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) was also significantly improved, suggesting that LmSTT3D has broad substrate specificity. These results suggest that the glycosylation status of recombinant proteins can be improved by heterologous STT3 expression, which will allow for the customization of therapeutic protein profiles.     

Scanning N-glycosylation mutagenesis of membrane proteins

N-Glycosylation of eukaryotic membrane proteins is a co-translational event that occurs in the lumen of the endoplasmic reticulum (ER). This process is catalyzed by a membrane-associated oligosaccharyl transferase (OST) complex that transfers a preformed oligosaccharide (Glc₃Man₉GlcNAc₂-) to an asparagine (Asn) side-chain acceptor located within the sequon (-Asn-X-Ser/Thr-). Scanning N-glycosylation mutagenesis experiments, where novel acceptor sites are introduced at unique sites within membrane proteins, have shown that the acceptor sites must be located a minimum distance (12–14 amino acids) away from the luminal membrane surface of the ER in order to be efficiently N-glycosylated. Scanning N-glycosylation mutagenesis can therefore be used to determine membrane protein topology and it can also serve as a molecular ruler to define the ends of transmembrane (TM) segments. Furthermore, since N-glycosylation is a co-translational event, N-glycosylation mutagenesis can be used to identify folding intermediates in membrane proteins that may expose segments to the ER lumen transiently during biosynthesis.     

Coordination of N-glycosylation and protein translocation across the endoplasmic reticulum membrane by Sss1 protein

Secretory proteins are translocated across the endoplasmic reticulum (ER) membrane through a channel formed by three proteins, namely Sec61p, Sbh1p, and Sss1p (Johnson, A. E., and van Waes, M. A. (1999) Annu. Rev. Cell Dev. Biol. 15, 799-842). Sec61p and Sss1p are essential for translocation (Esnault, Y., Blondel, M. O., Deshaies, R. J., Schekman, R., and Kepes, F. (1993) EMBO J. 12, 4083-4093). Sec61p is a polytopic membrane protein that lines the protein translocation channel. The role of Sss1p is unknown. During import into the ER through the Sec61p channel, many proteins are N-glycosylated before translocation is completed. In addition, both the Sec61 channel and oligosaccharyl transferase (OST) copurify with ribosomes from rough ER, suggesting that OST is located in close proximity to the Sec61 channel (Gorlich, D., Prehn, S., Hartmann, E., Kalies, K.-U., and Rapoport, T. A. (1992) Cell 71, 489-503 and Wang, L., and Dobberstein, B. (1999) FEBS Lett. 457, 316-322). Here, we demonstrate a direct interaction between Sss1p and a subunit of OST, Wbp1p, using the split-ubiquitin system and co-immunoprecipitation. We generated mutants in the cytoplasmic domain of Sss1p that disturb the interaction with OST and are viable but display a translocation defect specific for proteins with glycosylation acceptor sites. Our data suggest that Sss1p coordinates translocation across the ER membrane and N-linked glycosylation of secretory proteins.     

Identification and characterization of UDP-mannose in human cell lines and mouse organs: Differential distribution across brain regions and organs

Mannosylation in the endoplasmic reticulum is a key process for synthesizing various glycans. Guanosine diphosphate mannose (GDP-Man) and dolichol phosphate-mannose serve as donor substrates for mannosylation in mammals and are used in N-glycosylation, O-mannosylation, C-mannosylation, and the synthesis of glycosylphosphatidylinositol-anchor (GPI-anchor). Here, we report for the first time that low-abundant uridine diphosphate-mannose (UDP-Man), which can serve as potential donor substrate, exists in mammals. Liquid chromatography-mass spectrometry (LC-MS) analyses showed that mouse brain, especially hypothalamus and neocortex, contains higher concentrations of UDP-Man compared to other organs. In cultured human cell lines, addition of mannose in media increased UDP-Man concentrations in a dose-dependent manner. These findings indicate that in mammals the minor nucleotide sugar UDP-Man regulates glycosylation, especially mannosylation in specific organs or conditions.     

Pmt-mediated O mannosylation stabilizes an essential component of the secretory apparatus, Sec20p, in Candida albicans

Sec20p is an essential endoplasmic reticulum (ER) membrane protein in yeasts, functioning as a tSNARE component in retrograde vesicle traffic. We show that Sec20p in the human fungal pathogen Candida albicans is extensively O mannosylated by protein mannosyltransferases (Pmt proteins). Surprisingly, Sec20p occurs at wild-type levels in a pmt6 mutant but at very low levels in pmt1 and pmt4 mutants and also after replacement of specific Ser/Thr residues in the lumenal domain of Sec20p. Pulse-chase experiments revealed rapid degradation of unmodified Sec20p (38.6 kDa) following its biosynthesis, while the stable O-glycosylated form (50 kDa) was not formed in a pmt1 mutant. These results suggest a novel function of O mannosylation in eukaryotes, in that modification by specific Pmt proteins will prevent degradation of ER-resident membrane proteins via ER-associated degradation or a proteasome-independent pathway.     

Site Mapping and Characterization of O-Glycan Structures on α-Dystroglycan Isolated from Rabbit Skeletal Muscle

The main extracellular matrix binding component of the dystrophin-glycoprotein complex, α-dystroglycan (α-DG), which was originally isolated from rabbit skeletal muscle, is an extensively O-glycosylated protein. Previous studies have shown α-DG to be modified by both O-GalNAc- and O-mannose-initiated glycan structures. O-Mannosylation, which accounts for up to 30% of the reported O-linked structures in certain tissues, has been rarely observed on mammalian proteins. Mutations in multiple genes encoding defined or putative glycosyltransferases involved in O-mannosylation are causal for various forms of congenital muscular dystrophy. Here, we explore the glycosylation of purified rabbit skeletal muscle α-DG in detail. Using tandem mass spectrometry approaches, we identify 4 O-mannose-initiated and 17 O-GalNAc-initiated structures on α-DG isolated from rabbit skeletal muscle. Additionally, we demonstrate the use of tandem mass spectrometry-based workflows to directly analyze glycopeptides generated from the purified protein. By combining glycomics and tandem mass spectrometry analysis of 91 glycopeptides from α-DG, we were able to assign 21 different residues as being modified by O-glycosylation with differing degrees of microheterogeneity; 9 sites of O-mannosylation and 14 sites of O-GalNAcylation were observed with only two sites definitively exhibiting occupancy by either type of glycan. The distribution of identified sites of O-mannosylation suggests a limited role for local primary sequence in dictating sites of attachment.     

Functional characterization of extracellular chitinase encoded by the YlCTS1 gene in a dimorphic yeast Yarrowia lipolytica

The hemiascomycetes yeast Yarrowia lipolytica is a dimorphic yeast with alternating yeast and mycelia forms. Bioinformatic analysis revealed the presence of three putative chitinase genes, YlCTS1, YlCTS2, and YlCTS3, in the Y. lipolytica genome. Here, we demonstrated that the protein of YlCTS1 (YlCts1p), which contains an N-terminal secretion signal peptide, a long C-terminal Ser/Thr-rich domain, and a chitin-binding domain, is a homologue to Saccharomyces cerevisiae chitinase 1 (ScCts1p). Deletion of YlCTS1 remarkably reduced extracellular endochitinase activity in the culture supernatant of Y. lipolytica and enhanced cell aggregation, suggesting a role of YlCts1p in cell separation as ScCts1p does in S. cerevisiae. However, loss of YlCts1p function did not affect hyphal formation induced by fetal bovine serum addition. The mass of YlCts1p was dramatically decreased by jack bean α-mannosidase digestion but not by PNGase F treatment, indicating that YlCts1p is modified only by O-mannosylation without N-glycosylation. Moreover, the O-glycan profile of YlCts1p was identical to that of total cell wall mannoproteins, supporting the notion that YlCts1p can be used as a good model for studying O-glycosylation in this dimorphic yeast.     

Mechanism of Bacterial Oligosaccharyltransferase: IN VITRO QUANTIFICATION OF SEQUON BINDING AND CATALYSIS*

N-Linked glycosylation is an essential post-translational protein modification in the eukaryotic cell. The initial transfer of an oligosaccharide from a lipid carrier onto asparagine residues within a consensus sequon is catalyzed by oligosaccharyltransferase (OST). The first X-ray structure of a complete bacterial OST enzyme, Campylobacter lari PglB, was recently determined. To understand the mechanism of PglB, we have quantified sequon binding and glycosylation turnover in vitro using purified enzyme and fluorescently labeled, synthetic peptide substrates. Using fluorescence anisotropy, we determined a dissociation constant of 1.0 μm and a strict requirement for divalent metal ions for consensus (DQNAT) sequon binding. Using in-gel fluorescence detection, we quantified exceedingly low glycosylation rates that remained undetected using in vivo assays. We found that an alanine in the −2 sequon position, converting the bacterial sequon to a eukaryotic one, resulted in strongly lowered sequon binding, with in vitro turnover reduced 50,000-fold. A threonine is preferred over serine in the +2 sequon position, reflected by a 4-fold higher affinity and a 1.2-fold higher glycosylation rate. The interaction of the +2 sequon position with PglB is modulated by isoleucine 572. Our study demonstrates an intricate interplay of peptide and metal binding as the first step of protein N-glycosylation.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Molecular Mechanism of Substrate Specificity for Heparan Sulfate 2-O-Sulfotransferase

Heparan sulfate (HS) is an abundant polysaccharide in the animal kingdom with essential physiological functions. HS is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple enzymes. In vivo regulation of this process remains unclear. HS 2-O-sulfotransferase (2OST) is a key enzyme in this pathway. Here, we report the crystal structure of the ternary complex of 2OST, 3′-phosphoadenosine 5′-phosphate, and a heptasaccharide substrate. Utilizing site-directed mutagenesis and specific oligosaccharide substrate sequences, we probed the molecular basis of specificity and 2OST position in the ordered HS biosynthesis pathway. These studies revealed that Arg-80, Lys-350, and Arg-190 of 2OST interact with the N-sulfo groups near the modification site, consistent with the dependence of 2OST on N-sulfation. In contrast, 6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation. Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.