Identification of β-Secretase (BACE1) Substrates Using Quantitative Proteomics
Figure 4
BACE1 shedding of type I transmembrane proteins.
Identified BACE1 substrates of type I topology were either cloned and stably expressed or the endogenous protein was analyzed in HEK cells by Western blot. Cell lysates are shown in the left column, and conditioned medium shown in the right column. (A) LRIG2 was expressed in cell lysates as several distinct bands, likely owing to differential glycosylation (left panel). LRIG2 shedding by BACE1 was observed in the conditioned medium (right panel). (B) LRIG3 was stably expressed, as shown in cell lysates (left panel). LRIG3 was shed by BACE1 into the conditioned medium (right panel). (C) Endogenous IGF2R was analyzed with an ectodomain directed antibody. β-secretase activity produced a prominent decline in the full-length protein (left panel), and an increase in the shed ectodomain in the conditioned medium (right panel). (D) Endogenous APLP1 was expressed at undetectable levels in the cell lysate (left panel; the asterisk denotes a background band) but accumulated in the conditioned medium due to BACE1-mediated ectodomain shedding (right panel).