15.12
Proteinmodifikationen im RER
Die Modifikation sekretorischer und transmembraner Proteine, die in das raue ER gelangen, beginnt im ER-Lumen. Diese Modifikationen unterstützen die P…
Die N-verknüpfte Glykosylierung und die Bildung von Disulfidbindungen sind zwei wichtige Proteinmodifikationen, die im ER auftreten.
Während der Glykosylierung fügt der Oligosaccharyltransferase-Komplex verzweigte Oligosaccharidmoleküle zu einigen ausgewählten Asparaginresten einer eingehenden Polypeptidkette hinzu.
Im Gegensatz dazu findet die Bildung von Disulfidbindungen zwischen zwei eng beieinander liegenden Cysteinresten auf derselben oder unterschiedlichen Polypeptidketten statt.
Es sind zwei Hauptakteure beteiligt – die Proteindisulfid-Isomerase (PDI) und die ER-Oxidoreduktase I oder Ero1.
Das PDI-Molekül ähnelt einem Hufeisen mit Disulfidbindungen in seinem aktiven Zentrum.
Oxidiertes PDI hat eine offene Konformation und bindet ungefaltete Polypeptide. Zunächst bildet der reduzierte Cysteinrest des Polypeptids eine Disulfidbindung mit dem aktiven Zentrum des Enzyms.
Dieses Zwischenprodukt interagiert dann mit einem anderen eng beieinander liegenden Cystein auf der Polypeptidkette, was zu einer Disulfidbindung zwischen den beiden Resten führt.
Anschließend wechselt der PDI in eine geschlossene Konformation, wodurch das Polypeptid freigesetzt wird.
Ero1 wandelt den reduzierten PDI wieder in seinen oxidierten Zustand um und bereitet ihn auf eine weitere Reaktionsrunde vor.
View the full transcript and gain access to JoVE Core videos
Q1: What is N-linked glycosylation and why does it occur in the ER?
N-linked glycosylation is the covalent attachment of branched oligosaccharide molecules to asparagine residues on incoming polypeptide chains, catalyzed by the oligosaccharyltransferase complex. This modification improves protein folding kinetics, increases stability by masking cleavage sites, and allows ER chaperones to assess proper protein folding before the protein exits the ER.
Q2: How does protein disulfide isomerase facilitate disulfide bond formation?
Protein disulfide isomerase (PDI) resembles a horseshoe with disulfide bonds in its active site. Oxidized PDI binds unfolded polypeptides and forms a disulfide link between a reduced cysteine on the polypeptide and the enzyme's active site. This intermediate then interacts with another nearby cysteine, creating a disulfide bond between the two residues before PDI releases the polypeptide.
Q3: What role does Ero1 play in maintaining the ER redox environment?
ER oxidoreductase 1 (Ero1) recycles reduced protein disulfide isomerase back to its oxidized state, preparing it for additional rounds of disulfide bond formation. Ero1 utilizes a significant fraction of molecular oxygen available in the cell and generates hydrogen peroxide, maintaining redox homeostasis inside the ER while supporting oxidative protein folding.
Q4: Which amino acid sequences are recognized for N-linked glycosylation?
N-linked glycosylation occurs at asparagine residues within the tripeptide sequences Asn-X-Ser and Asn-X-Thr, where X represents any amino acid except proline. The oligosaccharyltransferase complex recognizes these specific motifs and adds oligosaccharides during both cotranslational and post-translational protein translocation in the ER lumen.
Q5: How does PDI function as both an enzyme and a proofreader for disulfide bonds?
Oxidized PDI catalyzes disulfide bond formation between cysteine residues, while reduced PDI acts as a proofreader by correcting inappropriately paired cysteines through rearranging disulfide linkages. This dual function ensures accurate disulfide bond formation and proper protein folding in the oxidizing ER environment.
Q6: What physical properties of proteins are altered by N-linked glycosylation?
N-linked glycosylation improves the thermodynamic kinetics of protein folding, allowing glycosylated proteins to fold better than their non-glycosylated counterparts. Glycosylation also increases protein stability by masking hydrophobic stretches and cleavage sites, protecting the protein from degradation and enhancing its structural integrity.
Q7: Where do disulfide bonds predominantly form in the cell?
Disulfide bonds form predominantly in the rough ER lumen, which provides an oxidizing environment favorable for their formation. A small fraction of disulfide bonds can also form in the mitochondrial intermembrane space, but the ER remains the primary site for this modification of secretory and transmembrane proteins.