Ufd1 Enhances The Activity Of Ubiquitin Ligase Gp78

Cholesterol is the most abundant sterol in mammalian cells. It mainly functions in regulating the fluidity and phase transitions of membranes. Cholesterol is maintained at a steady level in physiological conditions. High level of cholesterol often results in many diseases, such as atherosclerosis, gallstone and coronary heart disease. Therefore, animals have developed precise mechanisms to control cholesterol metabolism. Cholesterol synthesis is an important part in its metabolism and it is regulated by feedback inhibition. 3-hydroxy-3-methlglutaryl coenzyme A reductase (HMG CoA reductase, HMGCR), which is regulated by transcription and protein degradation, catalyzes the reduction of HMG CoA to mevalonate, a rate-limiting step in the synthesis of cholesterol. Sterols simulated proteasomal degradation of HMGCR is one of the regulatory pathway that cells employ to feedback control the synthesis of cholesterol. Gp78, which is a membrane-anchored ubiquitin ligase and interacts with Insig, catalyses the ubiquitination and degradation of HMGCR. The precursor of cholesterol, lanosterol, can directly stimulate the interaction between HMGCR and Insig-gp78. Hence, this interaction facilitates gp78 to catalyse the ubiquination reaction of HMGCR, and then, HMGCR is rapidly degraded through ER-associated protein degradation (ERAD). To further understand the mechanism of ER-associated degradation of HMG CoA reductase catalyzed by gp78, we utilize coimmunoprecipitation coupled with mass Spectrometry to identify gp78-interacting proteins and we found Ufd1. Next, their binding sites has been mapped by coimmunoprecipitation and GST-pull down assays. Further, we provide evidence to show that Ufd1 directly interacts with gp78 and enhances its E3 activity through their interactions. Meanwhile, we have identified that the mono-ubiquitin binding of Ufd1 is required for the enhancement of gp78 activity and poly-ubiquitin biding is critical for a post-ubiquitination step in ERAD. Our results illustrate that Ufd1, as a cofactor of gp78, performs dual functions in the sterol-regulated degradation of reductase and plays a critical role in ER-associated protein degradation and cholesterol metabolism.