Conditional Knockdown of Proteasomes Results in Cell-cycle Arrest and Enhanced Expression of Molecular Chaperones Hsp70 and Hsp40 in Chicken DT40 Cells

Abstract The 26 S proteasome is an evolutionarily conserved ATP-dependent protease complex that degrades poly-ubiquitinated proteins and plays essential roles in a critical part of cellular regulation. In vertebrates, the roles of the proteasome have been widely studied by use of specific inhibitors, but not genetically. Here, we generated a cell line Z−/−/−/Z-HA, in which the expression of the catalytic subunit of the proteasome, Z (β2) could be manipulated. This cell line expresses exogenous Z protein under the control of a tetracycline-repressible promoter in a Z-nullizygous genetic background. Treatment of these cells with doxycycline inhibited Z expression and, hence, the function of the proteasome. The latter resulted in accumulation of poly-ubiquitinated proteins and concomitant induction of molecular chaperones Hsp70 and Hsp40. These results suggest a synergistic role for the proteasome with these molecular chaperones to eliminate misfolded or damaged proteins in vivo. Furthermore, knockdown of the proteasome induced apoptotic cell death following cell-cycle arrest at G2/M phase. Our Z−/−/−/Z-HA cell line would be useful for evaluating proteolytic processes catalyzed by the proteasome in many biological events in vertebrate cells.