Yeast Genomics in the Elucidation of Endoplasmic Reticulum (ER) Quality Control and Associated Protein Degradation (ERQD)

Abstract

The endoplasmic reticulum (ER) is the eukaryotic organelle where most secreted proteins enter the secretory pathway. They enter this organelle in an unfolded state and are folded by a highly active folding machinery to reach their native state. The ER contains an efficient protein quality control system, which recognizes malfolded and orphan proteins and targets them for elimination by a mechanism called ER-associated degradation (ERAD). Both processes are tightly linked, and they will be abbreviated as ERQD (ER quality control and associated degradation). Because ERQD is highly conserved from yeast to man, the easy amenability of yeast to genetic and molecular biological studies combined with the knowledge of its genome and proteome makes it a preferred organism to study such "housekeeping" functions of eukaryotic cells. New genomic and proteomic methods have led to new experimental concepts. Genome-wide screens using genomic deletion libraries led to the identification of genes involved in the processes in question. Using such a genome-wide approach, we devise a sensitive growth test for selection of yeast mutants defective in ERQD. A chimeric protein (CTL*) was generated consisting of the ER luminal, N-glycosylated CPY* protein fused to a transmembrane domain and cytoplasmic 3-isopropylmalate dehydrogenase, the Leu2 protein. In addition, the nonglycosylated ER-membrane-located ERQD substrate Sec61-2p was fused to Leu2p (Sec61-2-L*). Cells carrying a LEU2 deletion can only grow on medium lacking leucine when the chimeric protein CTL* or Sec61-2-L* is not degraded. Thus, only mutant cells defective in an ERQD component can grow. A genome-wide screen can be performed by transforming the CTL* or Sec61-2-L* coding DNA into the approximately 5000 individual deletion mutants of the EUROSCARF yeast library. Examples for new components required for ERQD found by this method are the mannose-6-phosphate receptor domain protein Yos9p and the ubiquitin domain proteins Dsk2p and Rad23p.