Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC

Abstract

The 72-kDa zinc finger transcription factor PacC, distantly related to Ci/Gli developmental regulators, undergoes two-step proteolytic processing in response to alkaline ambient pH. "Signaling protease" cleavage of PacC72 removes a processing-inhibitory C-terminal domain, making its truncated PacC53 product accessible to a second " processing" protease, yielding PacC27. Features of the processing proteolysis suggested the proteasome as a candidate protease. We constructed, using gene replacements, two missense active site mutations in preB, the Aspergillus nidulans orthologue of Saccharomyces cerevisiae PRE2 encoding the proteasome β5 subunit. preB1K101A is lethal. Viable preB2K101R impairs growth and, like its equivalent pre2 K108R in yeast, impairs chymotryptic activity. pre2K108R and preB2K101R active site mutations consistently shift position of the scissile bonds when PacC is processed in S. cerevisiae and A. nidulans, respectively, indicating that PacC must be a direct substrate of the proteasome. preB2K101R leads to a 2-3-fold elevation in NimE mitotic cyclin levels but appears to result in PacC instability, suggesting an altered balance between processing and degradation. preB2K101R compensates the marked impairment in PacC27 formation resulting from deletion of the processing efficiency determinant in PacC, further indicating direct proteasomal involvement in the formation of PacC27. Deletion of a Gly-Pro-Ala-rich region within this processing efficiency determinant markedly destabilizes PacC. Arg substitutions of Lys residues within this efficiency determinant and nearby show that they cooperate to promote PacC processing. A quadruple Lys-to-Arg substitution (4K→R) impairs formation of PacC 27 and leads to persistence of PacC53. Wild-type PacC 53 becomes multiply phosphorylated upon alkaline pH exposure. Processing-impaired 4K→R PacC53 becomes excessively phosphorylated. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.