Figure 3
Non-canonical proTGFα secretion is mediated by its cytoplasmic tail and not the juxtamembrane region.
(a) Deletion of carboxy-terminal PDZ-binding domain (Δ4) reduces secretion of proTGFα, whereas deletion of the entire cytoplasmic tail (Δ31) completely prevents its release. (b) Secretion of proTGFα triggered by RHBDL4 ectopically expressed in Hek293T cells is unaffected by mutations of putative rhomboid-like cleavage sites in the juxtamembrane and adjacent TM region of FLAG-proTGFα. (c) Mutation of A93F and A93P do not prevent RHBDL4-induced secretion of proTGFα and secretion of a 17-kDa proTGFα fragment (indicated by asterisk) is independent of RHBDL4 activity in HCT116 cells. (d) RHBDL4-triggered non-canonical proTGFα secretion does not depend on membrane integral features shown by expression of a chimeric molecule harboring the TM domain of the stable ER protein calnexin (proTGFα-CNXTMD). (e) The active site S144A mutant of RHBDL4-GFP (SA) co-immunoprecipitates the ERAD substrate pTα but not proTGFα. Mutation of L274A and L278A (mt) in the conserved ubiquitin interacting motif of RHBDL4 reduce binding of pTα as has been observed previously17. IP, immunoprecipitation; WB, western blot. (f) Carboxy-terminal tail of proTGFα is sufficient for non-canonical secretion as demonstrated by the chimeric molecule CNX-TGFαtail.
