Cells adapted to the proteasome inhibitor 4-hydroxy5-iodo-3-nitrophenylacetyl-Leu-Leu-leucinal-vinyl sulfone require enzymatically active proteasomesfor continued survival

The proteasome is the primary protease used by cells for degrading proteins and generating peptide ligands for class I molecules of the major histocom patibility complex, Based on the properties of cells adapted to grow in the presence of the proteasome inhibitor 4-hydroxy-5-iodo-3-nitrophenylacetyl- Leu-Leu-leucinal-vinyl sulfone (NLVS), it was proposed that proteasomes can be replaced by alternative proteolytic systems, particularly a large prote olytic complex with a tripeptidyl peptidase II activity. Here we show that NLVS-adapted cells retain sensitivity to a number of highly specific protea some inhibitors with regard to antigenic peptide generation, accumulation o f polyubiquitinated proteins, degradation of p53, and cell viability. In ad dition, we show that in the same assays (with a single minor exception), NL VS-adapted cells are about as sensitive as nonselected cells to Ala-Ala-Phe -chloromethylketone, a specific inhibitor of tripeptidyl peptidase II activ ity. Based on these findings, we conclude that proteasomes still have essen tial proteolytic functions in adapted cells that are not replaced by Ala-Al a-Phe-chloromethylketone-sensitive proteases.