Endoplasmic Reticulum-Associated Protein Quality Control and Degradation: Genome-Wide Screen for ERAD Components
互联网
491
In this chapter, a genetic approach is presented that leads to the isolation of mutants and to the identification of proteins involved in protein quality control and endoplasmic reticulum-associated degradation (ERAD). The method makes use of a genomic screen of a yeast deletion library (EUROSCARF). Transformation of each of the approx 5000 strains deleted in one nonvital gene each with a CPundefined chimera (CTundefined, Fig. 1 ) containing CPundefined C-terminally fused to a transmembrane domain and the cytosolic Leu2 protein (3-isopropylmalate dehydrogenase) constitutes the basic screening procedure. Because of a Leu2p deficiency in all deletion strains, cells can grow only when the CTundefined chimera is present. As the CPundefined module of CTundefined will be recognized in ERAD-proficient cells, CTundefined will be degraded and the strain is unable to grow. Therefore the absence of genes necessary for ER quality control and ERAD will allow cell growth and indicate the necessity of the respective gene for these processes ( 1 ).
Fig. 1. Schematic representation of the ERAD substrates CPundefined and CTundefined. The mutated CPundefined remains malfolded in the ER lumen. The membrane protein CTundefined is a chimeric derivative of CPundefined, fused to a transmembrane domain and to Leu2p.
