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 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 ␤5 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.

Ambient pH signalling in the yeast Yarrowia lipolytica involves YlRim23p/PalC, which interacts with Snf7p/Vps32p, but does not require the long C terminus of YlRim9p/PalI

“…In Candida albicans, the genes encoding the three proteases Sap4p, Sap5p and Sap6p are under the control of extracellular pH, Sap5p being required for E-cadherin degradation during mucosal tissue invasion (Villar et al, 2007). This pathway has been intensively investigated in Aspergillus nidulans (Penalva & Arst, 2002; Herranz et al, 2005;Hervas-Aguilar et al, 2007; Galindo et al, 2007;Penas et al, 2007;Calcagno-Pizarelli et al, 2007), in Y. lipolytica (Lambert et al, 1997; Tréton et al, 2000;Gonzalez-Lopez et al, 2002;Blanchin-Roland et al, 2005), in several other ascomycetes, including Saccharomyces cerevisiae (Li & Mitchell, 1997;Xu & Mitchell, 2001;Vincent et al, 2003;Xu et al, 2004;Boysen & Mitchell, 2006), in C. albicans (Ramon & Fonzi, 2003;Li et al, 2004;Kullas et al, 2004;Cornet et al, 2005;Barwell et al, 2005;Baek et al, 2006), in the basidiomycete Ustilago maydis (Arechiga-Carvajal & Ruiz-Herrera, 2005) and in other fungi (Penalva & Arst, 2002. At alkaline pH, a cascade of six pal or RIM genes activates the zincfinger transcriptional factor PacC/Rim101p through a complex C-terminal proteolytic processing event (see below).…”

The transcription repressor NmrA is subject to proteolysis by three Aspergillus nidulans proteases

“…23 However, this differs from the case of NmrA where proteolysis negates the activity of a transcription repressor, in that PacC is processed to produce the biologically active form of a transcription activator. 23 Proteolytic processing of a transcription repressor has been reported in Human, and the possibility of regulating transcription factors by target-specific proteolysis has been suggested as a general mechanism. 24 For example, activation of the nuclear factor j-B (NF-j-B) is modulated by its interaction with the repressor protein IjB-a, and the activity of IjB-a is itself controlled by ubiquitination and subsequent proteasome-mediated degradation.…”

“…For example, the A. nidulans zinc finger-containing transcription factor PacC undergoes two step proteolytic processing in response to alkaline ambient pH. 23 However, this differs from the case of NmrA where proteolysis negates the activity of a transcription repressor, in that PacC is processed to produce the biologically active form of a transcription activator. 23 Proteolytic processing of a transcription repressor has been reported in Human, and the possibility of regulating transcription factors by target-specific proteolysis has been suggested as a general mechanism.…”

Pho85 Kinase, a Cyclin-Dependent Kinase, Regulates Nuclear Accumulation of the Rim101 Transcription Factor in the Stress Response of Saccharomyces cerevisiae

“…3A), phosphorylation of Rim101 would enhance its interaction with the exportin and conversely reduce its interaction with importin, the latter also contributing to the exclusion of Rim101 from the nucleus. A. nidulans PacC is also phosphorylated (9). In this case, however, it is speculated that phosphorylation leads to recruitment of the proteasome machinery that carries out the processing of the transcription factor (9).…”