Dynamic association of proteasomal machinery with the centrosome

Although the number of pathologies known to arise from the inappropriate fo lding of proteins continues to grow, mechanisms underlying the recognition and ultimate disposition of misfolded polypeptides remain obscure. For exam ple, how and where such substrates are identified and processed is unknown. We report here the identification of a specific subcellular structure in w hich, under basal conditions, the 20S proteasome, the PA700 and PA28 (700- and 180-kD proteasome activator complexes, respectively), ubiquitin, Hsp70 and Hsp90 (70- and 90-kD heat shock protein, respectively) concentrate in H EK 293 and HeLa cells. The structure is perinuclear, surrounded by endoplas mic reticulum, adjacent to the Golgi, and colocalizes with gamma-tubulin, a n established centrosomal marker. Density gradient fractions containing pur ified centrosomes are enriched in proteasomal components and cell stress ch aperones. The centrosome-associated structure enlarges in response to inhib ition of proteasome activity and the level of misfolded proteins. For examp le, folding mutants of CFTR form large inclusions which arise from the cent rosome upon inhibition of proteasome activity. At high levels of misfolded protein, the structure not only expands but also extensively recruits the c ytosolic pools of ubiquitin, Hsp70, PA700, PA28, and the 20S proteasome. Th us, the centrosome may act as a scaffold, which concentrates and recruits t he systems which act as censors and modulators of the balance between foldi ng, aggregation, and degradation.