Elucidating a New Mode of Function for ERD2 (K/HDEL Receptor) at the cis-Golgi

The secretory pathway is a network of organelles interfacing the endoplasmic reticulum (ER), the plasma membrane and the lysosomes/vacuoles. The Golgi apparatus plays a central role here, sending and receiving membrane carriers to and from these locations. The accumulation of soluble proteins in the ER is dependent on the K/HDEL receptor (ER retention defective 2 or ERD2), originally thought to capture ligands at the Golgi and recycle between these two compartments. In 2018 this model was challenged for the first time after discovery that the C-terminal fluorescent ERD2 fusions displaying the typical dual ER-Golgi localisation were biologically inactive. In this project I have systematically explored multiple lines of experiments, each of which had the potential to either support or refute the biological relevance of ERD2 recycling. I show that Golgi-residency of ERD2 is not a plant-specific feature but also applies to human ERD2. Furthermore, I could demonstrate that ligand-induced ERD2 recycling to the ER is an artefact caused by mutating, deleting or masking of a C-terminal di-leucine motif that is acting as a Golgi retention signal. Next, I illustrated the sensitivity of the ERD2 C-terminus to much smaller epitope fusions and even single residues, re-enforcing the requirement for a native C-terminus to enable meaningful functional studies. Approaching the subject from a different perspective, I discovered a strict correlation between biological activity and Golgi residency. This was substantiated by two complementary experiments; first I could show that the introduction of a COPII ER export signal can overcome the loss of Golgi retention. Secondly, fusing an alternative cis-Golgi retention signal can re-activate ERD2 when its own Golgi-retention motif is compromised. The combined experiments show that ERD2 does not recycle like other sorting receptors and must rely on a novel mechanism to capture ligands and segregate them into retrograde transport carriers without joining. To help dissect the as yet unknown sequence of events occurring upon ligand-binding and release at the cis-Golgi, I explored the subdomain structure of ERD2 and created dominant negative constructs which directly interfere with endogenous ERD2 function, strongly inducing the secretion of HDEL cargo. Whilst open ended, the tools generated will help elucidate the underlying mechanism conserved between plantae, mammals and protozoa that control the first and probably most ancient Golgi-mediated cargo sorting event in eukaryotes.