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 assumption that they are dispersed over the nucleoplasm and cytoplasm in all eukaryotic cells, we wanted to determine the subcellular distribution of 26S proteasomes in living yeast cells. For this purpose, we generated yeast strains that express functional green fluorescent protein (GFP) fusions of proteasomal subunits. An α 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/YOL145w, 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 yeast. These findings were supported by the co‐localization and co‐enrichment of 26S proteasomes with NE–ER marker proteins. A major location of proteasomal peptide cleavage activity was visualized in the NE–ER network, indicating that proteasomal degradation takes place mainly in this subcellular compartment in yeast.