Extended Data Fig. 8: RHBDL4 cleaves other ER-resident proteins before ER retention motifs and facilitates the secretion of N-terminal proteolytic fragments.

RHBDL4 cleavage assays for V5-tagged (a) PDIA6, (b) Calreticulin and (c) ERP44. The putative cleavage sites are indicated by blue arrows in the schematic representations. (a) WT RHBDL4 cleaved PDIA6 at the C-terminus, resulting in secretion of the N-terminal fragment (blue arrow) into the medium. (b) WT RHBDL4 cleaved Calreticulin at the internal region (minor cleavage) and the C-terminus (major cleavage), resulting in secretion of the N-terminal fragments (blue arrows) into the SN. (c) WT RHBDL4 cleaved ERP44 before RDEL sequence. The secreted proteins were deglycosylation mix II sensitive. Because we could not source an antibody to specifically detect the RDEL sequence, we inserted an HA-tag four amino acids before the RDEL sequence for detection. Anti-V5 detected proteolytic fragments 1 and 2 (p1 and p2), and p1 was also detected by the anti-HA antibody, indicating that one cleavage might happen after the HA-tag. The expression of RHBDL4 was detected by an anti-Strep antibody. RHBDL4 cleavage assays for (d) endogenous PDIA6 and (e) endogenous Calreticulin. (d) WT RHBDL4 cleaved endogenous PDIA6. The proteolytic fragment of PDIA6 (red arrow) without the KDEL sequence was secreted into the SN. (e) WT RHBDL4 cleaved endogenous Calreticulin. The proteolytic fragment of Calreticulin (red arrow) without the KDEL sequence was secreted into the SN. Expression of RHBDL4 did not increase the endogenous level of PDIA6 and Calreticulin (lanes 2 and 3 vs 1). The expression of RHBDL4 is shown in Fig. 3g. BFA inhibitory assays for (f) PDIA6 and (g) Calreticulin. The secretion of proteolytic fragments of PDIA6 and Calreticulin was inhibited compared to cells treated with DMSO (lanes 5 vs 2, 11 vs 8). Endogenous PDIA6 and Calreticulin were detected by an anti-PDIA6 and an anti-CALR antibody, respectively. The expression of RHBDL4 is shown in Extended Data Fig. 7a. (h) Endogenous RHBDL4 cleaves endogenous Calreticulin. WT RHBDL4-dependent Calreticulin proteolytic fragments in the extracellular media were detected by an anti-CALR antibody in WT HCT116 cells (red arrows), but not in RHBDL4 KO HCT116 cells. The secretion of the major proteolytic fragment (near full-length Calreticulin, low intensity) and the minor proteolytic fragment (high intensity) were both detected. Transferrin: loading control. The endogenous RHBDL4 is shown in Extended Data Fig. 7b. (a-h) were repeated in two biological replicates with similar results. For gel source data, see Supplementary Fig. 1.