For all living systems, there is a requirement to recycle and regulate proteins. In eukaryotic organisms this is accomplished by the proteasome. The p97 ATPase is another highly conserved and essential complex present throughout the eukaryotic cell. In Paper I we utilized UFD fluorescent substrates to address the role of p97 and cofactors in soluble proteasome degradation. Results using RNAi and Drosophila p97 mutants propose p97 to function upstream of the proteasome on cytosolic proteasome targets as an important unfoldase together with its Ufd1/Npl4 cofactors. The results implicate p97 to be important for degradation of proteasome substrates lacking natural extended peptide regions. In Paper II we focused on identifying transcription factors essential for production of proteasomal subunits and associated proteins in Drosophila S2 cells. We utilized an RNA library targeting 993 known or candidate transcription factors and monitored RNAi depleted Drosophila S2 cells expressing the UFD reporter UbG76VGFP. We identified a range of potential candidates and focused on the bZIP transcription factor Cnc-C. RNAi and qrt-PCR experiments implicated Cnc-C to be involved in transcription of proteasomal subunits. In Paper III we applied our knowledge gained from Paper I about p97 dependent substrates and set up a high-throughput microscopy screening method to potentially find inhibitors specifically targeting the p97 proteasomal sub-pathway. Utilizing UFD substrates with and without C-terminal peptide tails we determined if compounds inhibited the core proteasomal machinery or the p97 pathway specifically. Through a primary and secondary round of screening we identified several new compounds inhibiting the ubiquitin-proteasome pathway though none from our initial screening had specificity for p97.

A majority of intracellular proteins are degraded by the ubiquitin proteasome system (UPS). In this thesis, both the mechanism of the degradation and the regulation of UPS have been investigated. The importance of the p97 ATPase for proteasomal degradation of cytosolic substrates was examined. It was shown that tightly folded model substrates were dependent on p97 for their degradation. In addition to this, it was shown that an extended flexible peptide sequence on the substrate allowed degradation to occur directly by the proteasome. We propose that p97 works as an unfoldase on substrates that lack initiation regions. These results were originally achieved with experiments using Drosophila melanogaster S2 cell culture. Corresponding experiments were carried out in human cell lines. We observed that the human proteasome also needed assistance from the human p97 protein complex when model substrates lacked unfolded tagged regions. To identify the transcription factor(s) that regulate the expression of proteasomal genes, a large scale RNAi screen was performed. A library consisting of dsRNA to all known and predicted transcription factors in Drosophila was used. Drosophila S2 cells expressing the cytosolic Ub^G76V-GFP substrate were used in the screen. Since thisfusion protein isdependent on the UPS for its degradation,failure in UPS can easily be detected viafluorescent stabilization.When dsRNA targeted the bZIP transcription factor Cnc-C,it lead to a reduction of the proteasome subunit protein levels as well as decreased mRNA levels. Phylogenetic analysis together with sequence alignments were used to learn how Cnc-C is related to the bZIP CNC genes in other metazoans and in particular mammalian cells. In mammalian cells, NF-E2, Nrf1, Nrf2 and Nrf3 are present and we propose that Cnc-C is related to a common ancestor transcription factor for all these four genes. This contradicts earlier studies proposing that Cnc-C is a homolog of the mammalian Nrf2 protein.In the last study, theproteasome recovery pathway was examined tounderstand which bZIPtranscription factor in human cells is responsible for the expression of proteasome genes after proteasome inhibition.Different cancer cell lines were used to examine theexpression level of proteasome genes after treating the cells with proteasome inhibitors when either the bZIP protein Nrf1 or Nrf2 wereknocked down. It was shown that Nrf1-/- cellslacked the ability toupregulate proteasome genes after proteasomeinhibition. In contrast, Nrf2-/- cells still had the capacity to restore proteasome levels. This lead to the conclusion thatNrf1 is responsible for the proteasome recovery pathway in mammalian cells.