The Characterization of human Hrd3p/Sel1p

・Introduction
　　　　Protein biosynthesis is a surprisingly inefficient process, and approximately one-third of newly synthesized proteins are in some way faulty and are targeted for degradation during or soon after their synthesis. Because the accumulation and aggregation of such non-native proteins is detrimental to cells, the accuracy of protein synthesis and folding is strictly monitored by quality control (QC) system. The best-characterized QC system is the one in the endoplasmic reticulum (ER). Those proteins that cannot pass the QC system in ER are degraded through a system termed ER associated degradation (ERAD).
　　　　Studies in yeast have identified a number of proteins that are required for ERAD. One of these is called Hrd3p. Sel1p is the closest homolog of Hrd3p in mammalian cells and very little is known about this protein. The aims of this project were to clone and characterize Sel1p and gain information on the endogenous expression of Sel1p in various mammalian cells.

・Material and Methods
　　　　In this project, in vitro expressed radioactive Sel1p was used for characterization such as N-linked glycosylation, reduction and protease protection. Two types of cell lines (Cos-7 cells and Hela cells) and dog microsome solution were used to examine the endogenous Sel1p expression. This endogenous Sel1p was detected by an anti-sel1 antibody.

・Results and Discussion
　　　　From the characterization of Sel1p, we conclude that Sel1p is a membrane glycoprotein, and it probably contains at least one disulfide bond that is involved in maintaining its native conformation. In addition, Sel1p is likely to orient in lumen.
　　　　The anti-sel1 antibody specifically recognizes human and dog Sel1p. However, this antibody didn’t recognize monkey Sel1p. The failure to detect any Sel1p in Cos-7 cells could be due to a difference in the sequence of the monkey protein, or may reflect a low level of expression of the endogenous protein in these cells.


・Future work
　　　　To obtain more information on the expression of Sel1p, western blotting of different tissues such as brain, liver and kidney is needed. To demonstrate the localization of Sel1p in the cells, subcellular fractionation has to be done. In addition, it is also important to figure out whether or not Sel1p is involved in mammalian ERAD, by running native gels or using cross-linking regents to see if it forms larger complexes with the key proteins of mammalian ERAD.