Accurately measuring the ability of the K/HDEL receptor (ERD2) to retain the ER cargo Amy-HDEL has questioned earlier results on which the popular receptor recycling model is based upon. Here we demonstrate that ERD2 Golgi-retention, rather than fast ER export supports its function. Ligand-induced ERD2 redistribution is only observed when the C-terminus is masked or mutated, compromising the signal that prevents Golgi-to-ER transport of the receptor. Forcing COPI mediated retrograde transport destroys receptor function, but introducing ER-to-Golgi export or cis-Golgi retention signals re-activate ERD2 when its endogenous Golgi-retention signal is masked or deleted. We propose that ERD2 remains fixed as a Golgi gatekeeper, capturing K/HDEL proteins when they arrive and releasing them again into a subdomain for retrograde transport back to the ER. An in vivo ligand:receptor ratio far greater than 100 to 1 strongly supports this model, and the underlying mechanism appears to be extremely conserved across kingdoms.

The K/HDEL receptor (ER retention defective 2 or ERD2) does not recycle between compartments when sorting ER chaperones, contrary to the favoured model. A conserved C-terminal di-leucine motif specifically prevents ERD2 Golgi-to-ER transport and is not required for ER export. The Golgi-retention mechanism strips Golgi-membranes of the GTPase ARF1 so that ERD2 avoids accompanying its ligands in retrograde transport. When this motif is deleted or masked, introducing a fast ER-to-Golgi export signal or an alternative cis-Golgi retention signal re-activates ERD2. Meanwhile, forcing retrograde transport renders the receptor non-functional. We have established an in vivo ligand/receptor ratio far greater than 100 to 1, and propose a gatekeeper model to explain how few receptors at the Golgi can prevent the secretion of highly abundant soluble ER proteins. The underlying mechanism is conserved across kingdoms and will yield valuable insight into Golgi-mediated cargo sorting and cisternal compartment maintenance.