[Mapping of Rpn4p regions responsible for transcriptional activation of proteasome genes].

Rpn4p is positive and negative transcriptional regulator of the ubiquitin-proteasome system. At the same time, it is the extremely short-lived proteasome-associated protein and the proteasome substrate. Proteasome-dependent mechanisms of Rpn4p degradation is studied in details, but mechanisms of its action are not clear, and, first of all, functional domains is not defined. To map Rpn4p functionally important regions, we created a set of its deletion derivatives. Mutant proteins were expressed in rpn4-A yeast strain and their activities were estimated by measuring the mRNA level of proteasomal genes in appropriate transformants. We showed that Rpn4p devoid of its C-terminal region containing the zinc finger DNA-binding domains and its N-terminus, which shares no homology with transactivation domains of known transcription factors, was completely unable to activate transcription. Of two clusters of acid residues, proposed to be activation domains, only second one takes part in transcription. Moreover, deletion of N-terminus, proximal acidic domain or, surprisingly, zinc finger domains leads to Rpn4p stabilization. These data give new insight onto both mechanisms of Rpn4p action and degradation.

[Isolation and identification of PACE-binding protein rpn4--a new transcription activator, participating in regulation of 26S proteosome and other genes]. / Vydelenie i identifikatsiia PACE-sviazyvaiushchego belka Rpn4--novogo transkriptsionnogo aktivatora, uchastvuiushchego v reguliatsii 26S proteasomykh i drugikh genov.

A new upstream activating sequence (UAS) 5'-GGTGGCAAA-3' was detected in the promoters of 27 out of the 33 proteasomal genes and of several genes related to the ubiquitin-proteasomal system of Saccharomices cerevisiae. The sequence was termed proteasome-associated control element (PACE). Gel retardation assay revealed specific binding of PACE with an extracted protein. The protein (64K) was purified by affinity chromatography and was homogeneous by SDS-PAGE. Microsequencing showed that the protein is Rpn4p. The ability of Rpn4p to activate transcription was demonstrated with constructs containing fragments of the RPN5 and CDC48 gene promoters and reporter cat. Binding to PACE, Rpn4p may act as a common transcription factor on the proteasomal and proteasome-related genes.