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 candidate protease. We constructed, using gene replacements, two missense active site mutations in preB, the A. nidulans orthologue of Saccharomyces cerevisiae PRE2 encoding the proteasome beta5 subunit. preB1K101A is lethal. Viable preB2K101R impairs growth and like its equivalent pre2K108R in yeast impairs chymotryptic activity. pre2K108R and preB2K101R active site mutations consistently shift position of the scissile bonds when PacC is processed in Saccharomyces 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 altered balance between processing and degradation. preB2K101R compensates the marked impairment in PacC27 formation resulting from deletion of the processing efficiency determinant (PED) in PacC, further indicating direct proteasomal involvement in the formation of PacC27. Deletion of a Gly-Pro-Ala rich region within this PED 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 PacC27 and leads to persistence of PacC53. Wild-type PacC53 becomes multiply phosphorylated on alkaline pH exposure. Processing-impaired 4K>R PacC53 becomes excesively phosphorylated.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|