Rhomboid intramembrane proteases are the enzymes that release active epidermal growth factor receptor (EGFR) ligands in Drosophila and C. elegans, but little is known about their functions in mammals. Here we show that the mammalian rhomboid protease RHBDL4 (also known as Rhbdd1) promotes trafficking of several membrane proteins, including the EGFR ligand TGFα, from the endoplasmic reticulum (ER) to the Golgi apparatus, thereby triggering their secretion by extracellular microvesicles. Our data also demonstrate that RHBDL4-dependent trafficking control is regulated by G-protein coupled receptors, suggesting a role for this rhomboid protease in pathological conditions, including EGFR signaling. We propose that RHBDL4 reorganizes trafficking events within the early secretory pathway in response to GPCR signaling. Our work identifies RHBDL4 as a rheostat that tunes secretion dynamics and abundance of specific membrane protein cargoes.

Background: O-Mannosylation is a conserved, essential protein modification that is initiated in the endoplasmic reticulum (ER). Results: Protein O-mannosyltransferases act at the translocon complex to modify proteins entering the ER. Conclusion: Protein translocation into the ER, O-mannosylation, and N-glycosylation are coordinated processes. Significance: Defining the mechanism of O-mannosylation is crucial to understand its diverse physiological roles for ER homeostasis.