Evidence for the direct involvement of the proteasome in the proteolytic processing of the Aspergillus nidulans zinc finger transcription factor PacC.
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 PacC(72) removes a processing-inhibitory C-terminal domain, making its truncated PacC(53) product accessible to a second "processing" protease, yielding PacC(27). 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 beta5 subunit. preB1(K101A) is lethal. Viable preB2(K101R) impairs growth and, like its equivalent pre2(K108R) in yeast, impairs chymotryptic activity. pre2(K108R) and preB2(K101R) 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. preB2(K101R) 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. preB2(K101R) compensates the marked impairment in PacC(27) formation resulting from deletion of the processing efficiency determinant in PacC, further indicating direct proteasomal involvement in the formation of PacC(27). 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 PacC(53). Wild-type PacC(53) becomes multiply phosphorylated upon alkaline pH exposure. Processing-impaired 4K-->R PacC(53) becomes excessively phosphorylated.
Hervás-Aguilar
América
A
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid 28040, Spain.
Rodríguez
José M
JM
Tilburn
Joan
J
Arst
Herbert N
HN
Peñalva
Miguel A
MA
D000483:Alleles; D000595:Amino Acid Sequence; D001233:Aspergillus nidulans / Q000378:metabolism*; D005656:Fungal Proteins / Q000378:metabolism*; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D010641:Phenotype; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D016335:Zinc Fingers
D005656:Fungal Proteins; C092394:PacC protein, Aspergillus; D014157:Transcription Factors; D046988:Proteasome Endopeptidase Complex