The mechanism of post-ER organelle associated degradation

The function of all cells depends on the integrity of their proteome. Typical proteomes of eukaryotic cells are estimated to consist of 50 x 106 proteins in S. cerevisiae or 2 x 109 proteins in human cells. Among those over tens of millions of proteins, cellular quality control networks detect and selectively degrade only proteins that mis-fold, cannot integrate into protein complexes, fail to target to the correct organelles or are destined for degradation by regulatory mechanism. Two selective pathways are known to degrade membrane proteins in eukaryotic cells. The endoplasmic reticulum (ER) associated degradation (ERAD) pathway ubiquitinates membrane proteins at the ER and retro- translocates them into the cytoplasm for proteasomal degradation. Upon export from the ER, all other ubiquitinated membrane proteins are thought to be sorted exclusively by the endosomal sorting complexes required for transport (ESCRT) into the lumen of lysosomes for degradation. Using genetic screens, we now identified in S. cerevisiae a third pathway for the degradation of membrane proteins: It is a post-ER organelle-associated degradation pathway (pERAD) that selectively targets membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. An endogenous substrate of pERAD is the ER-resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis, whose over-expression is associated with diabetes, ulcerative colitis, Crohns disease and asthma. Phosphorylation of Orm2 triggers ER export to Golgi and endosomes, where Orm2 is then poly-ubiquitinated by the membrane embedded Defective in SREBP cleavage (Dsc) ubiquitin ligase complex. Ubiquitination of Orm2 and the function of the AAA-ATPase Cdc48/VCP are essential for its proteasomal degradation (Schmidt, O. et al. submitted) Our discovery of pERAD as the third membrane protein degradation pathway and its role in protein and membrane homeostasis provides a starting point to address the following questions in this research project: (1) How are the substrates of pERAD selected by the Dsc complex (2) how is the pERAD pathway linked with cellular stress response pathways and quality control networks and (3) how does the pERAD pathway mechanistically enable the proteasomal degradation of membrane proteins from post-ER compartments. To address these questions we will use a combination of yeast genetics, quantitative proteomics, biochemical approaches and live cell imaging. We expect that the results of this research project will provide a detailed conceptual and mechanistic framework for pERAD in yeast, plant and humans.

Our project reveals a new molecular mechanism that maintains the subcellular organization of cells, with the Golgi apparatus (or Golgi for short) playing a crucial role. It was already known that the Golgi acts as a central sorting station for proteins and lipids. Our work now unveils that the Golgi also serves as a critical hub for membrane quality control. In this regard, we have discovered the molecular mechanism involved. At the Golgi, there is a ubiquitin ligase complex (known as the Dsc complex) capable of recognizing whether proteins belong to this organelle or not. Proteins that have strayed or are orphaned are identified and selectively destroyed. Consequently, the Dsc complex prevents the spreading of orphaned proteins from the Golgi to other organelles, thereby preserving the cellular protein and lipid composition. We are confident that our findings will advance the understanding of cellular quality control - a crucial area in pathology and biology in general.