scholarly journals A Cytosolic Reductase Pathway is Required for Complete N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protein Translocation ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
Translation System ◽  
Acceptor Site ◽  
Post Translational Modification

AbstractN-linked glycosylation of proteins entering the secretory pathway is an essential post-translational modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide bond, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is dependent on the STT3A-containing oligosaccharide transferase. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

scholarly journals A Cytosolic Reductase Pathway is Required for Efficient N-Glycosylation of an STT3B-Dependent Acceptor Site

2021 ◽  
Author(s):  
Marcel van Lith ◽  
Marie Anne Pringle ◽  
Bethany Fleming ◽  
Giorgia Gaeta ◽  
Jisu Im ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Site Occupancy ◽  
Secretory Protein ◽  
Glycosylation Site ◽  
Redox Conditions ◽  
In Vitro Translation ◽  
Translation System ◽  
Acceptor Site ◽  
Reductive Pathway

N-linked glycosylation of proteins entering the secretory pathway is an essential modification required for protein stability and function. Previously, it has been shown that there is a temporal relationship between protein folding and glycosylation, which influences the occupancy of specific glycosylation sites. Here we use an in vitro translation system that reproduces the initial stages of secretory protein translocation, folding and glycosylation under defined redox conditions. We found that the efficiency of glycosylation of hemopexin was dependent upon a robust NADPH-dependent cytosolic reductive pathway, which could also be mimicked by the addition of a membrane impermeable reducing agent. The identified hypoglycosylated acceptor site is adjacent to a cysteine involved in a short range disulfide, which has been shown to be dependent on the STT3B-containing oligosaccharyl transferase. We also show that efficient glycosylation at this site is influenced by the cytosolic reductive pathway acting on both STT3A and STT3B-dependent glycosylation. Our results provide further insight into the important role of the ER redox conditions in glycosylation site occupancy and demonstrate a link between redox conditions in the cytosol and glycosylation efficiency.

scholarly journals Secretory protein translocation in a yeast cell-free system can occur posttranslationally and requires ATP hydrolysis.

1986 ◽  
Vol 102 (5) ◽  
pp. 1543-1550 ◽  
Author(s):  
M G Waters ◽  
G Blobel
Keyword(s):  
Atp Hydrolysis ◽  
Protein Translocation ◽  
Secretory Protein ◽  
Translation System ◽  
Yeast Saccharomyces Cerevisiae ◽  
Free System ◽  
In Vitro System ◽  
Alpha Factor ◽  
Microsomal Membranes

We describe an in vitro system with all components derived from the yeast Saccharomyces cerevisiae that can translocate a yeast secretory protein across microsomal membranes. In vitro transcribed prepro-alpha-factor mRNA served to program a membrane-depleted yeast translation system. Translocation and core glycosylation of prepro-alpha-factor were observed when yeast microsomal membranes were added during or after translation. A membrane potential is not required for translocation. However, ATP is required for translocation and nonhydrolyzable analogues of ATP cannot serve as a substitute. These findings suggest that ATP hydrolysis may supply the energy required for translocation of proteins across the endoplasmic reticulum.

scholarly journals Post-translational integration and oligomerization of connexin 26 in plasma membranes and evidence of formation of membrane pores: implications for the assembly of gap junctions

2002 ◽  
Vol 365 (3) ◽  
pp. 693-699 ◽  
Author(s):  
Shoeb AHMAD ◽  
W. Howard EVANS
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Translation System ◽  
Alternative Mechanism ◽  
Protein Cleavage ◽  
Membrane Components ◽  
Gap Junction Hemichannels

Gap-junction channels provide a widespread intercellular signalling mechanism. They are constructed of a family of connexin membrane proteins that thread across the membrane four times and oligomerize to generate hexameric gap-junction hemichannels. Using an in vitro cell-free transcription/translation system, we demonstrate that connexin (Cx) 26, one of the smallest connexins, is integrated directly in a post-translational manner into plasma membranes. Protein-cleavage studies of Cx26 integrated into plasma membranes indicate a similar native transmembrane topography to that of Cx26 integrated co-translationally into microsomes. Cx26 integrated post-translationally into plasma membranes oligomerizes and, when incorporated into liposomes, provides permeability to ascorbic acid, suggesting that gap-junction hemichannels are generated. The results provide the basis of a novel alternative mechanism for spontaneous assembly in plasma membranes of Cx26 gap-junction hemichannels that occurs independently of the conventional biogenesis of gap junctions involving connexin trafficking and oligomerization via membrane components of the secretory pathway.

scholarly journals Quantitative glycoproteomics reveals new classes of STT3A- and STT3B-dependent N-glycosylation sites

2019 ◽  
Vol 218 (8) ◽  
pp. 2782-2796 ◽  
Author(s):  
Natalia A. Cherepanova ◽  
Sergey V. Venev ◽  
John D. Leszyk ◽  
Scott A. Shaffer ◽  
Reid Gilmore
Keyword(s):  
Protein Translocation ◽  
Site Occupancy ◽  
Glycosylation Site ◽  
Wild Type ◽  
New Class ◽  
Cysteine Rich Protein ◽  
Glycosylation Sites ◽  
Flanking Sequences ◽  
Dependent Site ◽  
Mutant Cells

Human cells express two oligosaccharyltransferase complexes (STT3A and STT3B) with partially overlapping functions. The STT3A complex interacts directly with the protein translocation channel to mediate cotranslational glycosylation, while the STT3B complex can catalyze posttranslocational glycosylation. We used a quantitative glycoproteomics procedure to compare glycosylation of roughly 1,000 acceptor sites in wild type and mutant cells. Analysis of site occupancy data disclosed several new classes of STT3A-dependent acceptor sites including those with suboptimal flanking sequences and sites located within cysteine-rich protein domains. Acceptor sites located in short loops of multi-spanning membrane proteins represent a new class of STT3B-dependent site. Remarkably, the lumenal ER chaperone GRP94 was hyperglycosylated in STT3A-deficient cells, bearing glycans on five silent sites in addition to the normal glycosylation site. GRP94 was also hyperglycosylated in wild-type cells treated with ER stress inducers including thapsigargin, dithiothreitol, and NGI-1.

scholarly journals Characterization of a component of the yeast secretion machinery: identification of the SEC18 gene product.

1988 ◽  
Vol 8 (10) ◽  
pp. 4098-4109 ◽  
Author(s):  
K A Eakle ◽  
M Bernstein ◽  
S D Emr
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Yeast Cells ◽  
In Vitro Translation ◽  
Cell Fractionation ◽  
Significant Similarity

SEC18 gene function is required for secretory protein transport between the endoplasmic reticulum (ER) and the Golgi complex. We cloned the SEC18 gene by complementation of the sec18-1 mutation. Gene disruption has shown that SEC18 is essential for yeast cell growth. Sequence analysis of the gene revealed a 2,271-base-pair open reading frame which could code for a protein of 83.9 kilodaltons. The predicted protein sequence showed no significant similarity to other known protein sequences. In vitro transcription and translation of SEC18 led to the synthesis of two proteins of approximately 84 and 82 kilodaltons. Antisera raised against a Sec18-beta-galactosidase fusion protein also detected two proteins (collectively referred to as Sec18p) in extracts of 35S-labeled yeast cells identical in size to those seen by in vitro translation. Mapping of the 5' end of the SEC18 mRNA revealed only one major start site for transcription, which indicates that the multiple forms of Sec18p do not arise from mRNAs with different 5' ends. Results of pulse-chase experiments indicated that the two forms of Sec18p are not the result of posttranslational processing. We suggest that translation initiating at different in-frame AUG start codons is likely to account for the presence of two forms of Sec18p. Hydrophobicity analysis indicated that the proteins were hydrophilic in nature and lacked any region that would be predicted to serve as a signal sequence or transmembrane anchor. Although potential sites for N-linked glycosylation were present in the Sec18p sequence, the sizes of the in vivo SEC18 gene products were unaffected by the drug tunicamycin, indicating that Sec18p does not enter the secretory pathway. These results suggest that Sec18p resides in the cell cytoplasm. While preliminary cell fractionation studies showed that Sec18p is not associated with the ER or Golgi complex, association with a 100,000 x g pellet fraction was observed. This suggests that Sec18p may bind transiently to small vesicles such as those presumed to participate in secretory protein transport between ER and the Golgi complex.

Insights into the Secretory Pathway, the Mechanism of Terminal Glycosylation and Intracellular Peptide Hormone Receptors from Studies on Hepatic Golgi Fractions

1981 ◽  
Vol 39 ◽  
pp. 516-519
Author(s):  
J.J.M. Bergeron ◽  
B.I. Posner ◽  
Jacques Paiement ◽  
R. Sikstrom ◽  
M. Khan
Keyword(s):  
Golgi Apparatus ◽  
Plasma Proteins ◽  
Secretory Pathway ◽  
Hormone Receptors ◽  
Peptide Hormone ◽  
Secretory Protein ◽  
Golgi Membrane ◽  
Membrane Structures

Recent studies on purified subcellular fractions of hepatic Golgi apparatus have provided insight into the functioning of the Golgi apparatus in vivo.The hepatocyte is the site of synthesis of most circulating plasma proteins. On a total protein basis, purified Golgi fractions revealed mainly secretory content (albumin, transferrin and other plasma proteins) as major constituents. After an in vivo injection of radiolabeled leucine, newly synthesized secretory protein followed a temporal route from cis to trans regions of Golgi apparatus before appearance in the plasma. This route was revealed by studies on disrupted Golgi fractions enriched in disparate regions of the Golgi apparatus.The terminal glycosylation of secretory glcyoproteins (e.g. transferrin) can be studied by observing the transfer of UDP-(3H)-galactose to endogenous acceptors within Golgi fractions. Transfer was shown to occur to a glycolipid (dolichyl galactosyl phosphate) probably on the cytosolic aspect of the Golgi membrane. Translocation of the labeled galactose across the membrane coincided with fusion of Golgi saccules in vitro. It is felt that during the process of Golgi membrane fusion, inverted lipid- micellar membrane structures translocate the dolichyl galactosyl phosphate from a cytosolic to a luminal orientation. Luminally oriented dolichyl galactosyl phosphate would then serve as substrate for galactose transfer to intraluminal glycopeptide acceptors via intraluminal galactosyl transferase enzyme.

Neuroendocrine secretory protein 7B2: structure, expression and functions

2001 ◽  
Vol 357 (2) ◽  
pp. 329-342 ◽  
Author(s):  
Majambu MBIKAY ◽  
Nabil G. SEIDAH ◽  
Michel CHRÉTIEN
Keyword(s):  
Secretory Pathway ◽  
Secretory Granules ◽  
Secretory Protein ◽  
Neuroendocrine Cells ◽  
High Similarity ◽  
Granule Formation ◽  
Proteolytic Activation ◽  
Polyproline Motif

7B2 is an acidic protein residing in the secretory granules of neuroendocrine cells. Its sequence has been elucidated in many phyla and species. It shows high similarity among mammals. A Pro-Pro-Asn-Pro-Cys-Pro polyproline motif is its most conserved feature, being carried by both vertebrate and invertebrate sequences. It is biosynthesized as a precursor protein that is cleaved into an N-terminal fragment and a C-terminal peptide. In neuroendocrine cells, 7B2 functions as a specific chaperone for the proprotein convertase (PC) 2. Through the sequence around its Pro-Pro-Asn-Pro-Cys-Pro motif, it binds to an inactive proPC2 and facilitates its transport from the endoplasmic reticulum to later compartments of the secretory pathway where the zymogen is proteolytically matured and activated. Its C-terminal peptide can inhibit PC2 in vitro and may contribute to keep the enzyme transiently inactive in vivo. The PC2–7B2 model defines a new neuroendocrine paradigm whereby proteolytic activation of prohormones and proneuropeptides in the secretory pathway is spatially and temporally regulated by the dynamics of interactions between converting enzymes and their binding proteins. Interestingly, unlike PC2-null mice, which are viable, 7B2-null mutants die early in life from Cushing's disease due to corticotropin (‘ACTH’) hypersecretion by the neurointermediate lobe, suggesting a possible involvement of 7B2 in secretory granule formation and in secretion regulation. The mechanism of this regulation is yet to be elucidated. 7B2has been shown to be a good marker of several neuroendocrine cell dysfunctions in humans. The possibility that anomalies in its structure and expression could be aetiological causes of some of these dysfunctions warrants investigation.

scholarly journals Ipomoeassin-F disrupts multiple aspects of secretory protein biogenesis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peristera Roboti ◽  
Sarah O’Keefe ◽  
Kwabena B. Duah ◽  
Wei Q. Shi ◽  
Stephen High
Keyword(s):  
Stress Responses ◽  
Secretory Pathway ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Unfolded Protein ◽  
Cellular Stress Responses ◽  
Protein Entry ◽  
Protein Response ◽  
Er Homeostasis

AbstractThe Sec61 complex translocates nascent polypeptides into and across the membrane of the endoplasmic reticulum (ER), providing access to the secretory pathway. In this study, we show that Ipomoeassin-F (Ipom-F), a selective inhibitor of protein entry into the ER lumen, blocks the in vitro translocation of certain secretory proteins and ER lumenal folding factors whilst barely affecting others such as albumin. The effects of Ipom-F on protein secretion from HepG2 cells are twofold: reduced ER translocation combined, in some cases, with defective ER lumenal folding. This latter issue is most likely a consequence of Ipom-F preventing the cell from replenishing its ER lumenal chaperones. Ipom-F treatment results in two cellular stress responses: firstly, an upregulation of stress-inducible cytosolic chaperones, Hsp70 and Hsp90; secondly, an atypical unfolded protein response (UPR) linked to the Ipom-F-mediated perturbation of ER function. Hence, although levels of spliced XBP1 and CHOP mRNA and ATF4 protein increase with Ipom-F, the accompanying increase in the levels of ER lumenal BiP and GRP94 seen with tunicamycin are not observed. In short, although Ipom-F reduces the biosynthetic load of newly synthesised secretory proteins entering the ER lumen, its effects on the UPR preclude the cell restoring ER homeostasis.

scholarly journals NovelN-Benzoyl-2-Hydroxybenzamide Disrupts Unique Parasite Secretory Pathway

2012 ◽  
Vol 56 (5) ◽  
pp. 2666-2682 ◽  
Author(s):  
Alina Fomovska ◽  
Qingqing Huang ◽  
Kamal El Bissati ◽  
Ernest J. Mui ◽  
William H. Witola ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Protozoan Parasite ◽  
Secretory Protein ◽  
Content Type ◽  
Apicomplexan Parasites ◽  
Dense Granules ◽  
Genome Wide ◽  
Nanomolar Range

ABSTRACTToxoplasma gondiiis a protozoan parasite that can damage the human brain and eyes. There are no curative medicines. Herein, we describe our discovery ofN-benzoyl-2-hydroxybenzamides as a class of compounds effective in the low nanomolar range againstT. gondii in vitroandin vivo. Our lead compound, QQ-437, displays robust activity against the parasite and could be useful as a new scaffold for development of novel and improved inhibitors ofT. gondii. Our genome-wide investigations reveal a specific mechanism of resistance toN-benzoyl-2-hydroxybenzamides mediated by adaptin-3β, a large protein from the secretory protein complex.N-Benzoyl-2-hydroxybenzamide-resistant clones have alterations of their secretory pathway, which traffics proteins to micronemes, rhoptries, dense granules, and acidocalcisomes/plant-like vacuole (PLVs).N-Benzoyl-2-hydroxybenzamide treatment also alters micronemes, rhoptries, the contents of dense granules, and, most markedly, acidocalcisomes/PLVs. Furthermore, QQ-437 is active against chloroquine-resistantPlasmodium falciparum. Our studies reveal a novel class of compounds that disrupts a unique secretory pathway ofT. gondii, with the potential to be used as scaffolds in the search for improved compounds to treat the devastating diseases caused by apicomplexan parasites.

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