scholarly journals The alpha subunit of the Saccharomyces cerevisiae oligosaccharyltransferase complex is essential for vegetative growth of yeast and is homologous to mammalian ribophorin I.

1995 ◽  
Vol 128 (4) ◽  
pp. 525-536 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
P J Rapiejko ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Vegetative Growth ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Alpha Subunit ◽  
Protein Sequence Analysis ◽  
Exogenous Lipid ◽  
Microsomal Membranes ◽  
High Mannose

Oligosaccharyltransferase mediates the transfer of a preassembled high mannose oligosaccharide from a lipid-linked oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six nonidentical subunits (alpha-zeta), two of which are glycoproteins (alpha and beta). The beta and delta subunits of the oligosaccharyltransferase are encoded by the WBP1 and SWP1 genes. Here we describe the functional characterization of the OST1 gene that encodes the alpha subunit of the oligosaccharyltransferase. Protein sequence analysis revealed a significant sequence identity between the Saccharomyces cerevisiae Ost1 protein and ribophorin I, a previously identified subunit of the mammalian oligosaccharyltransferase. A disruption of the OST1 locus was not tolerated in haploid yeast showing that expression of the Ost1 protein is essential for vegetative growth of yeast. An analysis of a series of conditional ost1 mutants demonstrated that defects in the Ost1 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins at both the permissive and restrictive growth temperatures. Microsomal membranes isolated from ost1 mutant yeast showed marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Microsomal membranes isolated from the ost1 mutants contained elevated amounts of the Kar2 stress-response protein.

scholarly journals The essential OST2 gene encodes the 16-kD subunit of the yeast oligosaccharyltransferase, a highly conserved protein expressed in diverse eukaryotic organisms.

1995 ◽  
Vol 131 (2) ◽  
pp. 371-383 ◽  
Author(s):  
S Silberstein ◽  
P G Collins ◽  
D J Kelleher ◽  
R Gilmore
Keyword(s):  
Protein Sequence ◽  
Apoptotic Cell ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Temperature Sensitive ◽  
Microsomal Membranes ◽  
High Mannose ◽  
Eukaryotic Organisms

Oligosaccharyltransferase catalyzes the transfer of a preassembled high mannose oligosaccharide from a dolichol-oligosaccharide donor to consensus glycosylation acceptor sites in newly synthesized proteins in the lumen of the rough endoplasmic reticulum. The Saccharomyces cerevisiae oligosaccharyltransferase is an oligomeric complex composed of six non-identical subunits (alpha-zeta). The alpha, beta, gamma, and delta subunits of the oligosaccharyltransferase are encoded by the OST1, WBP1, OST3, and SWP1 genes, respectively. Here we describe the functional characterization of the OST2 gene that encodes the epsilon-subunit of the oligosaccharyltransferase. Genomic disruption of the OST2 locus was lethal in haploid yeast showing that expression of the Ost2 protein is essential for viability. Overexpression of the Ost2 protein suppresses the temperature-sensitive phenotype of the wbp1-2 allele and increases in vivo and in vitro oligosaccharyltransferase activity in a wbp1-2 strain. An analysis of a series of conditional ost2 mutants demonstrated that defects in the Ost2 protein cause pleiotropic underglycosylation of soluble and membrane-bound glycoproteins. Microsomal membranes isolated from ost2 mutant yeast show marked reductions in the in vitro transfer of high mannose oligosaccharide from exogenous lipid-linked oligosaccharide to a glycosylation site acceptor tripeptide. Surprisingly, the Ost2 protein was found to be 40% identical to the DAD1 protein (defender against apoptotic cell death), a highly conserved protein initially identified in vertebrate organisms. The protein sequence of ost2 mutant alleles revealed mutations at highly conserved residues in the Ost2p/DAD1 protein sequence.

scholarly journals Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 326
Author(s):  
Danwei Yu ◽  
Yang Su ◽  
Xiaohui Ding ◽  
Yuanmei Zhu ◽  
Bo Qin ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Secondary Mutation ◽  
Fusion Inhibitors ◽  
Hiv 1

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

GLUT8, the enigmatic intracellular hexose transporter

2009 ◽  
Vol 296 (4) ◽  
pp. E614-E618 ◽  
Author(s):  
Stefan Schmidt ◽  
Hans-Georg Joost ◽  
Annette Schürmann
Keyword(s):  
Cellular Localization ◽  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Sperm Cells ◽  
Similar Distribution ◽  
Class Iii ◽  
Extracellular Loop ◽  
Microsomal Membranes ◽  
Heterologous Expression Systems

GLUT8 is a class III sugar transporter predominantly expressed in testis and brain. In contrast to the class I and class II transporters, hydrophobicity plots predict a short extracellular loop between transmembrane domain (TM)1 and TM2 and a long extracellular loop between TM9 and TM10 that contains the only N-glycosylation site. In vitro translated GLUT8 migrates as a 35-kDa protein that is glycosylated in the presence of microsomal membranes. In heterologous expression systems, glucose transport activity ( Km of 2 mM) was inhibited by fructose and galactose. The transporter carries an NH2-terminal endosomal/lysosomal targeting motif ([DE]XXXL[LI]). Accordingly, constitutive GLUT8 has been found to be associated with endosomes and lysosomes but also with membranes of the endoplasmic reticulum. A similar distribution was detected after overexpression of wild-type or tagged GLUT8 in different cell systems. In these cells, none of the conventional signals tested induced a translocation of GLUT8 to the plasma membrane. Therefore, GLUT8 appears to catalyze transport of sugars or sugar derivatives through intracellular membranes. Slc2a8 knockout mice were viable, developed normally, and showed mild alterations in brain (increased proliferation of neuronal cells in dentate gyrus of the hippocampus, hyperactivity), heart (impaired transmission of electrical wave through the atrium), and sperm cells (reduced number of motile sperm cells associated with reduced mitochondrial membrane potential and ATP levels in sperm). The links between molecular function, cellular localization and phenotype of the knockout mouse is unclear and remains to be determined.

scholarly journals Binding interactions control SNARE specificity in vivo

2008 ◽  
Vol 183 (6) ◽  
pp. 1089-1100 ◽  
Author(s):  
Hui-Ju Yang ◽  
Hideki Nakanishi ◽  
Song Liu ◽  
James A. McNew ◽  
Aaron M. Neiman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Vegetative Growth ◽  
Snare Complex ◽  
Mutant Form ◽  
Prospore Membrane ◽  
Complex Mutation ◽  
Ionic Layer

Saccharomyces cerevisiae contains two SNAP25 paralogues, Sec9 and Spo20, which mediate vesicle fusion at the plasma membrane and the prospore membrane, respectively. Fusion at the prospore membrane is sensitive to perturbation of the central ionic layer of the SNARE complex. Mutation of the central glutamine of the t-SNARE Sso1 impaired sporulation, but does not affect vegetative growth. Suppression of the sporulation defect of an sso1 mutant requires expression of a chimeric form of Spo20 carrying the SNARE helices of Sec9. Mutation of two residues in one SNARE domain of Spo20 to match those in Sec9 created a form of Spo20 that restores sporulation in the presence of the sso1 mutant and can replace SEC9 in vegetative cells. This mutant form of Spo20 displayed enhanced activity in in vitro fusion assays, as well as tighter binding to Sso1 and Snc2. These results demonstrate that differences within the SNARE helices can discriminate between closely related SNAREs for function in vivo.

scholarly journals Functional characterization of Ost3p. Loss of the 34-kD subunit of the Saccharomyces cerevisiae oligosaccharyltransferase results in biased underglycosylation of acceptor substrates.

1995 ◽  
Vol 130 (3) ◽  
pp. 567-577 ◽  
Author(s):  
D Karaoglu ◽  
D J Kelleher ◽  
R Gilmore
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Functional Characterization ◽  
Null Mutant ◽  
Wild Type ◽  
Membrane Glycoproteins ◽  
En Bloc ◽  
Oligosaccharide Moiety

Within the lumen of the rough endoplasmic reticulum, oligosaccharyltransferase catalyzes the en bloc transfer of a high mannose oligosaccharide moiety from the lipid-linked oligosaccharide donor to asparagine acceptor sites in nascent polypeptides. The Saccharomyces cerevisiae oligosaccharyltransferase was purified as a heteroligomeric complex consisting of six subunits (alpha-zeta) having apparent molecular masses of 64 kD (Ost1p), 45 kD (Wbp1p), 34 kD, 30 kD (Swp1p), 16 kD, and 9 kD. Here we report a structural and functional characterization of Ost3p which corresponds to the 34-kD gamma-subunit of the oligosaccharyltransferase. Unlike Ost1p, Wbp1p, and Swp1p, expression of Ost3p is not essential for viability of yeast. Instead, ost3 null mutant yeast grow at wild-type rates on solid or in liquid media irrespective of culture temperature. Nonetheless, detergent extracts prepared from ost3 null mutant membranes are twofold less active than extracts prepared from wild-type membranes in an in vitro oligosaccharyltransferase assay. Furthermore, loss of Ost3p is accompanied by significant underglycosylation of soluble and membrane-bound glycoproteins in vivo. Compared to the previously characterized ost1-1 mutant in the oligosaccharyltransferase, and the alg5 mutant in the oligosaccharide assembly pathway, ost3 null mutant yeast appear to be selectively impaired in the glycosylation of several membrane glycoproteins. The latter observation suggests that Ost3p may enhance oligosaccharide transfer in vivo to a subset of acceptor substrates.

scholarly journals ACAT1 and ACAT2 Membrane Topology Segregates a Serine Residue Essential for Activity to Opposite Sides of the Endoplasmic Reticulum Membrane

2000 ◽  
Vol 11 (11) ◽  
pp. 3675-3687 ◽  
Author(s):  
Charles W. Joyce ◽  
Gregory S. Shelness ◽  
Matthew A. Davis ◽  
Richard G. Lee ◽  
Kelly Skinner ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Glycosylation Site ◽  
Membrane Topology ◽  
Endoplasmic Reticulum Membrane ◽  
Flag Epitope ◽  
Particle Assembly ◽  
Ester Formation ◽  
Site Use ◽  
Microsomal Membranes

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser269) in the cytosol and the analogous residue in ACAT2 (Ser249) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

scholarly journals Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

2013 ◽  
Vol 24 (12) ◽  
pp. 2034-2044 ◽  
Author(s):  
Lars Boeckmann ◽  
Yoshimitsu Takahashi ◽  
Wei-Chun Au ◽  
Prashant K. Mishra ◽  
John S. Choy ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Histone H3 ◽  
In Vitro Assays ◽  
Histone H3 Variant

The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtubule attachment or reduced cohesion. We determine that evolutionarily conserved Ipl1/Aurora B contributes to phosphorylation of Cse4, as levels of phosphorylated Cse4 are reduced at centromeres in ipl1 strains in vivo, and in vitro assays show phosphorylation of Cse4 by Ipl1. Consistent with these results, we observe that a phosphomimetic cse4-4SD mutant suppresses the temperature-sensitive growth of ipl1-2 and Ipl1 substrate mutants dam1 spc34 and ndc80, which are defective for chromosome biorientation. Furthermore, cell biology approaches using a green fluorescent protein–labeled chromosome show that cse4-4SD suppresses chromosome segregation defects in dam1 spc34 strains. On the basis of these results, we propose that phosphorylation of Cse4 destabilizes defective kinetochores to promote biorientation and ensure faithful chromosome segregation. Taken together, our results provide a detailed analysis, in vivo and in vitro, of Cse4 phosphorylation and its role in promoting faithful chromosome segregation.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

The Influence of Carbohydrate Structure on the Clearance of Recombinant Tissue-Type Plasminogen Activator

1988 ◽  
Vol 60 (02) ◽  
pp. 255-261 ◽  
Author(s):  
A Hotchkiss ◽  
C J Refino ◽  
C K Leonard ◽  
J V O'Connor ◽  
C Crowley ◽  
...  
Keyword(s):  
Amino Acid ◽  
Plasminogen Activator ◽  
Sodium Periodate ◽  
Glycosylation Site ◽  
Tissue Type ◽  
Carbohydrate Structure ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
High Mannose ◽  
Oligosaccharide Structures

SummaryModification of the carbohydrate structures of recombinant tissue-type plasminogen activator (rt-PA) can increase or decrease its rate of clearance in rabbits. When rt-PA was treated with sodium periodate to oxidize carbohydrate residues, the rate of clearance was decreased from 9.6 ± 1.9 ml min−1 kg−1 to 3.5 ± 0.6 ml min−1 kg−1 (mean ± SD, n = 5). A similar change in the clearance of rt-PA was introduced by the use of endo-β-N-acetyl- glucosaminidase H (Endo-H), which selectively removes high mannose asparagine-linked oligosaccharides; the clearance of Endo-H-treated rt-PA was 5.0 ± 0.5 ml min−1 kg−1. A mutant of rt-PA was produced with an amino acid substitution at position 117 (Asn replaced with Gin) to remove a potential glycosylation site that normally contains a high mannose structure. The clearance of this material was also decreased, similar to the periodate and Endo-H-treated rt-PA. Conversely, when rt-PA was produced in the CHO 15B cell line, which can produce only high mannose oligosaccharide structures on glycoproteins, the clearance was increased by a factor of 1.8. These results demonstrate that the removal of rt-PA from the blood depends significantly upon the nature of its oligosaccharide structures.

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