RESUMO
In response to alkaline ambient pH, the Aspergillus nidulans PacC transcription factor mediating pH regulation of gene expression is activated by proteolytic removal of a negative-acting C-terminal domain. We demonstrate interactions involving the approximately 150 C-terminal PacC residues and two regions located immediately downstream of the DNA binding domain. Our data indicate two full-length PacC conformations whose relative amounts depend upon ambient pH: one 'open' and accessible for processing, the other 'closed' and inaccessible. The location of essential determinants for proteolytic processing within the two more upstream interacting regions probably explains why the interactions prevent processing, whereas the direct involvement of the C-terminal region in processing-preventing interactions explains why C-terminal truncating mutations result in alkalinity mimicry and pH-independent processing. A mutant PacC deficient in pH signal response and consequent processing behaves as though locked in the 'closed' form. Single-residue substitutions, obtained as mutations bypassing the need for pH signal transduction, identify crucial residues in each of the three interactive regions and overcome the processing deficiency in the 'permanently closed' mutant.
Assuntos
Proteínas Fúngicas , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Sítios de Ligação/genética , Endopeptidases/metabolismo , Regulação da Expressão Gênica , Genes Fúngicos , Concentração de Íons de Hidrogênio , Modelos Biológicos , Dados de Sequência Molecular , Mutação Puntual , Conformação Proteica , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Deleção de Sequência , Transdução de Sinais , Fatores de Transcrição/química , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Dedos de Zinco/genéticaRESUMO
Addition of Ca2+ (1 to 10 mM) to submerged cultures of Penicillium cyclopium induces conidiation. Ca2+ induced an increase in cytosolic pH from approximately 7.00 to > 7.60 in less than 10 min, as determined with the fluorescent pH probe fluorescein. Measurement of the H(+)-ATPase activity in total membrane fractions did not show any stable activation in vivo as a result of Ca2+ treatment. By fluorescence ratio imaging microscopy, it was observed that vegetative hyphae exhibit a tip-to-base pH gradient, with the tip being more acidic. Ca2+ caused this gradient to dissipate within 10 min. The effect of several agents that are supposed to cause internal acidification, by different means, on conidiation was tested. Concentrations of these agents that did not significantly affect growth but inhibited Ca(2+)-induced conidiation also prevented the intracellular alkalinization observed after exposure to the cation. Calcium channel blockers (lanthanum, cobalt, verapamil, and nifedipine) were not able to inhibit Ca(2+)-induced conidiation, although their effect on calcium uptake was not evaluated. However, the combined results point towards externally bound Ca2+ as the primary agent of conidiation induction, causing changes in plasma membrane function which disrupt the pH gradient observed during apical growth.
