SUBCELLULAR-DISTRIBUTION OF PROTEASOMES IMPLICATES A MAJOR LOCATION OF PROTEIN-DEGRADATION IN THE NUCLEAR-ENVELOPE ER NETWORK IN YEAST

26S proteasomes are the key enzyme complexes responsible for selective turnover of short-lived and misfolded proteins. Based on the assumpti on that they are dispersed over the nucleoplasm and cytoplasm in all e ukaryotic cells, we wanted to determine the subcellular distribution o f 26S proteasomes in living yeast cells. For this purpose, we generate d yeast strains that express functional green fluorescent protein (GFP ) fusions of proteasomal subunits, An a subunit of the proteolytically active 20S core complex of the 26S proteasome, Pre6/YOL038w, as well as an ATPase-type subunit of the regulatory 19S cap complex, Cim5/YOL1 45w, were tagged with GFP. Both chimeras were shown to be incorporated completely into active 26S proteasomes. Microscopic analysis revealed that GFP-labelled 20S as well as 19S subunits are accumulated mainly in the nuclear envelope (NE)-endoplasmic reticulum (ER) network in yea st, These findings mere supported by the co-localization and co-enrich ment of 26S proteasomes with NE-ER marker proteins. A major Location o f proteasomal peptide cleavage activity was visualized in the NE-ER ne twork, indicating that proteasomal degradation takes place mainly in t his subcellular compartment in yeast.