Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

Mueller, Susanne; Wåhlander, Åsa; Selevsek, Nathalie; Otto, Claudia; Ngwa, Elsy M.; Poljak, Kristina; Frey, Alexander D.; Aebi, Markus; Gauss, Robert

doi:10.1091/mbc.e15-03-0168

Cited by 51 publications

(73 citation statements)

References 56 publications

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“…Little is known about the precise function of Ost5, but this small protein links Stt3 to the other subcomplexes within the OTase multiprotein complex (20,35) Fractions were calculated as in Figs. 1 and 2.…”

Section: Resultsmentioning

confidence: 99%

“…As with Ost3 and Ost6, Ost5 is not a catalytic subunit of OTase, and it appears to be a small linker subunit tethering other OTase subunits. Ost5 appears to form a subcomplex with Ost1, and this subcomplex associates with the catalytic Stt3 subunit of OTase via Ost5 (20,35). Ost5 is required for optimal OTase function in vitro, but OST5 deletion has no effect on the stability of Ost1 or Stt3 subunits, and has been previously reported to cause only a minor hypoglycosylation phenotype in yeast (20,35).…”

Section: Discussionmentioning

confidence: 97%

“…Ost5 appears to form a subcomplex with Ost1, and this subcomplex associates with the catalytic Stt3 subunit of OTase via Ost5 (20,35). Ost5 is required for optimal OTase function in vitro, but OST5 deletion has no effect on the stability of Ost1 or Stt3 subunits, and has been previously reported to cause only a minor hypoglycosylation phenotype in yeast (20,35). Our results here suggest a role for Ost5 in determining some aspect of site-specific glycosylation by OTase, as not all sites were equally affected by its deletion.…”

Section: Discussionmentioning

confidence: 99%

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SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery

Zacchi

Schulz

2016

Molecular & Cellular Proteomics

138

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Glycan macro-and microheterogeneity have profound impacts on protein folding and function. This heterogeneity can be regulated by physiological or environmental factors. However, unregulated heterogeneity can lead to disease, and mutations in the glycosylation process cause a growing number of Congenital Disorders of Glycosylation. We systematically studied how mutations in the N-glycosylation pathway lead to defects in mature proteins using all viable Saccharomyces cerevisiae strains with deletions in genes encoding Endoplasmic Reticulum lumenal mannosyltransferases (Alg3, Alg9, and Alg12), glucosyltransferases (Alg6, Alg8, and Die2/Alg10), or oligosaccharyltransferase subunits (Ost3, Ost5, and Ost6). To measure the changes in glycan macro-and microheterogeneity in mature proteins caused by these mutations we developed a SWATH-mass spectrometry glycoproteomics workflow. We measured glycan structures and occupancy on mature cell wall glycoproteins, and relative protein abundance, in the different mutants. All mutants showed decreased glycan occupancy and altered cell wall proteomes compared with wild-type cells. Mutations in earlier mannosyltransferase or glucosyltransferase steps of glycan biosynthesis had stronger hypoglycosylation phenotypes, but glucosyltransferase defects were more severe. ER mannosyltransferase mutants displayed substantial global changes in glycan microheterogeneity consistent with truncations in the glycan transferred to protein in these strains. Although ER glucosyltransferase and oligosaccharyltransferase subunit mutants broadly showed no change in glycan structures, ost3⌬ cells had shorter glycan structures at some sites, consistent with increased protein quality control mannosidase processing in this severely hypoglycosylating mutant. This method allows facile relative quantitative glycoproteomics, and our results provide insights into global regulation of site-specific glycosylation. Protein glycosylation is a highly conserved co-and posttranslocational modification of proteins that influences protein folding, stability, solubility, and function (1, 2). N-glycosylation of Asparagine (Asn) 1 residues occurs in eukaryota, archaea, and some bacteria, although the biosynthetic pathways and glycan structures are diverse in these organisms (3). In eukaryotes, nascent polypeptides in the Endoplasmic Reticulum (ER) are the protein acceptor substrates for N-glycosylation, as folded proteins cannot be efficiently N-glycosylated and N-glycosylation is critical for efficient protein folding. After protein folding, glycan structures can be further truncated or extended by Golgi-resident glycosyltransferases. Glycan biosynthesis is inherently inefficient, resulting in structural diversity of mature glycoproteins. Diversity in the presence or absence of glycans on glycoproteins is termed macroheterogeneity, whereas diversity in the structures of glycans at a specific site is termed microheterogeneity. This structural diversity is key for the regulation of many biological functions of glycoproteins...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 97%

Section: Discussionmentioning

confidence: 99%

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SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery

Zacchi

Schulz

2016

Molecular & Cellular Proteomics

138

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“…These experiments showed a strongly decreased stability of the dimerization-defective p65 FL/DD protein in both cell systems, suggesting that free and monomeric p65 undergoes rapid degradation. This mechanism might be used to ensure the balanced subunit stoichiometry of NF-κB complexes and similar mechanisms occur in a number of multiprotein complexes (44, 45). …”

Section: Resultsmentioning

confidence: 99%

NF‐κB p65 dimerization and DNA‐binding is important for inflammatory gene expression

et al. 2018

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Increasing evidence shows that many transcription factors execute important biologic functions independent from their DNA-binding capacity. The NF-κB p65 (RELA) subunit is a central regulator of innate immunity. Here, we investigated the relative functional contribution of p65 DNA-binding and dimerization in p65-deficient human and murine cells reconstituted with single amino acid mutants preventing either DNA-binding (p65 E/I) or dimerization (p65 FL/DD). DNA-binding of p65 was required for RelB-dependent stabilization of the NF-κB p100 protein. The antiapoptotic function of p65 and expression of the majority of TNF-α–induced genes were dependent on p65’s ability to bind DNA and to dimerize. Chromatin immunoprecipitation with massively parallel DNA sequencing experiments revealed that impaired DNA-binding and dimerization strongly diminish the chromatin association of p65. However, there were also p65-independent TNF-α–inducible genes and a subgroup of p65 binding sites still allowed some residual chromatin association of the mutants. These sites were enriched in activator protein 1 (AP-1) binding motifs and showed increased chromatin accessibility and basal transcription. This suggests a mechanism of assisted p65 chromatin association that can be in part facilitated by chromatin priming and cooperativity with other transcription factors such as AP-1.—Riedlinger, T., Liefke, R., Meier-Soelch, J., Jurida, L., Nist, A., Stiewe, T., Kracht, M., Schmitz, M. L. NF-κB p65 dimerization and DNA-binding is important for inflammatory gene expression.

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“…The importance of the regulatory role of ERAD or of protein degradation in the control of cells or organisms homeostasis appears to clash with the observation that most proteins in Escherichia coli , Saccharomyces cerevisiae and Metazoa cells are extremely stable. Their abundance must therefore be controlled at the translational level and, in the case of supramolecular complexes, cells must know how many molecules of individual complex subunits are needed to eventually assemble the functional protein (Figure ). This is a nice way to spare energy by preventing the production of excessive amounts of subunits that must then be destroyed.…”

Section: Question 5: What Is Erad For?mentioning

confidence: 99%

Five Questions (with their Answers) on ER‐Associated Degradation

Pisoni

Molinari

2016

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Production of a functional proteome is a major burden for our cells.Native proteins operate inside and outside the cells to eventually warrant life and adaptation to metabolic and environmental changes, there is no doubt that production and inappropriate handling of misfolded proteins may cause severe disease states. This review focuses on protein destruction, which is, paradoxically, a crucial event for cell and organism survival. It regulates the physiological turnover of proteins and the clearance of faulty biosynthetic products. It mainly relies on the intervention of two catabolic machineries, the ubiquitin proteasome system and the (auto)lysosomal system. Here, we have selected five questions dealing with how, why and when proteins produced in the mammalian endoplasmic reticulum are eventually selected for destruction.

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Protein degradation corrects for imbalanced subunit stoichiometry in OST complex assembly

Cited by 51 publications

References 56 publications

SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery

SWATH-MS Glycoproteomics Reveals Consequences of Defects in the Glycosylation Machinery

NF‐κB p65 dimerization and DNA‐binding is important for inflammatory gene expression

Five Questions (with their Answers) on ER‐Associated Degradation

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