Studies On The Function And Degradation Of RPN4

The ubiquitin-proteasome system plays key regulatory roles in many aspects of cellular regulation, such as metabolic adaptation, cell differentiation, cell cycle control and removal of abnormal proteins. The 26S proteasome, the multisubunit enzyme complex responsible for the destruction of ubiquitin-protein conjugates and other proteins, appears to be present in all eukaryotic cells, its subunit composition being highly conserved among species.In 1999, G.Mannhaupt identified a new, unique upstream activating sequence (5'-GGTGGCAAA-3') in the promoters of 26 out of the 32 proteasomal yeast genes characterized to date, which is called proteasome-associated control element. By using the one-hybrid method, they show that the factor binding to the proteasome-associated control element is Rpn4p, a protein containing a C2H2-type finger motif and two acidic domains, whose /raw-activating capacity is well in agreement with its sequence characteristics.Early studies showed that Rpn4 (also named Son1 and Ufd5) is a transcriptional activator of the Saccharomyces cerevisiae proteasome genes, and that Rpn4 is rapidly degraded by the 26S proteasome. These observations suggested that in vivo proteasome abundance may be regulated by an Rpn4-dependent feedback circuit in which the Rpn4 protein level appears to be a key determinant.In this experiment, we present direct evidence to support the Rpn4-proteasome feedback model. First, Pulse-chase analysis showed that Rpn4 was stabilized in two 5. cerevisiae proteasome mutants, pre1-1 and cim5-1. pre1-1 is a temperature-sensitive (to) mutant with defective Pre1, a subunit of the 20S core . The cim5-1 mutant bears a ts mutation in Rpt1, an essential ATPase of the 19S particle. Second, We found that the level of Rpn1~F and Pre6~F, subunits of the 26S proteasome, were significantly higher in the prel-1 mutant than in the congenic wt strain. The higher steady-state levels of Pre6 and Rpn1 were not due to their stabilization in the proteasome mutants because these proteasome subunits are long-lived even in wt cells. In agreement with the increase in protein levels, Northern analysis showed that the mRNA levels of RPN1 and PRE6 were higher (~2-fold) in the pre1-1 and cim5-1 mutants than in the wt cells. Thus, the proteasome expression is up-regulated in the proteasome mutants.