Mapping crucial cysteine residues for SNO-BACE1. (A) Schematic diagram of BACE1. BACE1 is a 501 amino-acid transmembrane protein. Signal peptide (1-21 aa) and pro-peptide (22-45 aa) are cleaved during protein maturation. Within the lumenal domain there are two active site motifs (signature sequences of aspartyl proteases) situated at amino acids 93-96 and 289-292, four N-glycosylation sites and six cysteine residues involved in intramolecular disulfide bonds. The transmembrane domain is located between amino acids 461 and 477, and the C-terminal part of the protein (24 aa) is located in the cytosol. (B) Site-directed mutagenesis data on the Cys mutants of BACE1. SNO-BACE1 levels were determined by biotin-switch assays and data were analyzed by densitometry of the SNO-BACE1/total BACE1 ratio to identify crucial cysteine residues for s-nitrosylation of BACE1. Wild type and mutants of BACE1-HA expression plasmids were transfected into HEK293 cells. After biotin-switch assay, SNO-BACE1 and total BACE1 proteins were probed with an anti-HA antibody.