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. 2011 Apr 1;145(1):79–91. doi: 10.1016/j.cell.2011.02.047

Figure 5.

Figure 5

iRhom Inhibits Secretion of EGF Family Ligands

(A–C) COS7 cell supernatants (sup) were analyzed for Grk secretion, and cell extracts were blotted for levels of HA-tagged iRhom and Unc93B (Brinkmann et al., 2007). The ratios of iRhom to Drosophila Rhomboid-1 (R1) indicate relative amounts of transfected DNA.

(A) Rhomboid-1 (R1)-induced secretion of Flag-Grk was inhibited by increasing amounts of iRhom.

(B) Secretion of the FLAG-tagged Delta was unaffected by increasing amounts of iRhom.

(C) Overexpression of the ER resident polytopic membrane protein Unc93B did not interfere with Grk secretion.

(D) Mouse iRhom2 (iR2) inhibited mouse RHBDL2 (R2)-induced secretion of EGF. Secreted mouse EGF was detected by anti-EGF. The metalloprotease inhibitor BB94 suppressed nonspecific shedding of EGF. Increasing amounts of HA-tagged mouse iRhom2 (bottom) inhibited RHBDL2-mediated EGF secretion (top).

(E and F) Drosophila iRhom destabilizes intracellular Gurken. Rhomboid-1-mediated Grk processing was inhibited by iRhom (E), but not Unc93B (F). Histograms show relative substrate (S) to product (P) conversion from the western blots above. The iRhom-to-Rho1 (iRhom:R1) and Unc93B:R1 ratios indicate relative amounts of transfected DNA. Equal loading was confirmed by probing cell extracts for actin levels. Error bars represent mean ± SD.

(G) The iRhom effect was rescued by expression of ER-localized, but not Golgi-localized, active rhomboid. KDEL-tagged Rhomboid-1 (R1-KDEL), but not the inactive serine-to-alanine mutant Rhomboid-1 (Rho1 SA-KDEL), induced secretion of Grk in supernatants. A constant amount of iRhom inhibited Grk secretion (8× more iRhom DNA transfected than R1-KDEL). Increasing amounts of HA-tagged R1-KDEL rescued Grk secretion. Using the same approach, untagged Rhomboid-1, which is Golgi localized (see Figure 2D), did not rescue Grk secretion (right). HA-tagged iRhom, R1-KDEL, and R1 were detected in cell extracts. Flag-tagged Grk was detected in the supernatants (top) and extracts (middle). The Grk substrate band was absent when R1-KDEL is used because of efficient processing when substrate and enzyme are both in the ER.

See also Figure S2.