Analysis of transmembrane domain mutants is consistent with sequential cleavage of Notch by gamma-secretase.
gamma-Secretase is a lipid-embedded, intramembrane-cleaving aspartyl protease that cleaves its substrates twice within their transmembrane domains (TMD): once near the cytosolic leaflet (at S3/epsilon) and again in the middle of the TMD (at S4/gamma). To address whether this unusual process occurs in two independent or interdependent steps, we investigated how mutations at the S3/epsilon site in Notch1-based substrates impact proteolysis. We demonstrate that such mutations greatly inhibit not only gamma-secretase-mediated cleavage at S3 but also at S4, independent of their impact on NICD stability. These results, together with our previous observations, suggest that hydrolysis at the center of the Notch transmembrane domain (S4/gamma) is dependent on the S3/epsilon cleavage. Notch (and perhaps all gamma-secretase substrates) may be cleaved by sequential proteolysis starting at S3.
Chandu
Dilip
D
Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
Huppert
Stacey S
SS
Kopan
Raphael
R
D053829:Amyloid Precursor Protein Secretases; D016282:Aspartic Acid Endopeptidases; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D003600:Cytosol / Q000201:enzymology; D010450:Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D047468:Immunoprecipitation; D007527:Isoenzymes / Q000378:metabolism; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D009154:Mutation / Q000502:physiology; D010447:Peptide Hydrolases / Q000737:chemistry; D011480:Protease Inhibitors / Q000494:pharmacology; D051881:Receptor, Notch1 / Q000378:metabolism*
D007527:Isoenzymes; D011480:Protease Inhibitors; D051881:Receptor, Notch1; D008715:Methionine; D053829:Amyloid Precursor Protein Secretases; D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; C509280:BACE1 protein, human; D007930:Leucine; C012211:ubenimex