Academic journal article Genetics

Mutations in Genes Encoding Sorting Nexins Alter Production of Intracellular and Extracellular Proteases in Aspergillus Nidulans

Academic journal article Genetics

Mutations in Genes Encoding Sorting Nexins Alter Production of Intracellular and Extracellular Proteases in Aspergillus Nidulans

Article excerpt

ABSTRACT

XprG, a putative p53-like transcriptional activator, regulates production of extracellular proteases in response to nutrient limitation and may also have a role in programmed cell death. To identify genes that may be involved in the XprG regulatory pathway, xprG2 revertants were isolated and shown to carry mutations in genes which we have named sogA-C (suppressors of xprG). The translocation breakpoint in the sogA1 mutant was localized to a homolog of Saccharomyces cerevisiae VPS5 and mapping data indicated that sogB was tightly linked to a VPS17 homolog. Complementation of the sogA1 and sogB1 mutations and identification of nonsense mutations in the sogA2 and sogB1 alleles confirmed the identification. Vps17p and Vps5p are part of a complex involved in sorting of vacuolar proteins in yeast and regulation of cell-surface receptors in mammals. Protease zymograms indicate that mutations in sogA-C permit secretion of intracellular proteases, as in S. cerevisiae vps5 and vps17 mutants. In contrast to S. cerevisiae, the production of intracellular protease was much higher in the mutants. Analysis of serine protease gene expression suggests that an XprG-independent mechanism for regulation of extracellular protease gene expression in response to carbon starvation exists and is activated in the pseudorevertants.

THE extracellular proteases of the filamentous fungus Aspergillus nidulans can be used as a model to study the response to starvation as production of extracellular proteases is repressed in nutrient-sufficient growth medium and is stimulated by the absence of a carbon, nitrogen, or sulfur source, irrespective ofwhetherprotein is present (Cohen 1973a). The xprG gene plays a major role in the regulation of extracellular proteases. Loss-offunction mutations in xprG abolish the production of extracellular proteases in response to carbon starvation and the response to nitrogen and sulfur starvation is reduced (Katz et al. 2006). In addition, production of an acid phosphatase in response to phosphate starvation is lost in xprG^sup -^ mutants indicating that XprG may be involved in a general response to starvation (Katz et al. 2006). A single gain-of-function mutation in xprG has been identified. Strains carrying the xprG1 mutation have increased extracellular protease activity in response to carbon and, to a lesser extent, nitrogen starvation (Katz et al. 1996). The xprG1 mutation results in an amino acid substitution (R186W) in the putative DNA-binding domain of XprG.

XprG belongs to a newly identified group of proteins that contains an Ndt80-like DNA-binding domain and belongs to the family of p53-like transcription factors (pfam.sanger.ac.uk/). Ndt80 activates the transcription of .150 genes during middle meiosis in Saccharomyces cerevisiae (Chu et al. 1998). Nutrient limitation is required for the initiation and completion of meiosis in S. cerevisiae (Esposito and Klapholz 1981). The ndt80 mutants arrest at the point in meiosis past which progression through meiosis cannot be reversed by nutrient supplementation (Xu et al. 1995). Thus Ndt80 may, like XprG, be involved in sensing nutrient status (Katz et al. 2006). Mutations in xprG do not affect meiosis.

Genetic evidence suggests that two noncatalytic hexokinase-like proteins (HxkC and HxkD) are components of the XprG regulatory pathway (Katz et al. 2000; Bernardo et al. 2007). Loss-of-function mutations in hxkC and hxkD produce a similar phenotype to the xprG1 gain-of-function mutation and are suppressed by xprG^sup -^ mutations. HxkD is a nuclear protein whereas HxkC is associated with mitochondria (Bernardo et al. 2007). The binding of hexokinase to mitochondria blocks apoptosis in human tumor cells (Pastorino et al. 2002) and regulates programmed cell death in plants (Kim et al. 2006). The similarity between XprG and Vib-1, which is required for expression of genes during programmed cell death in Neurospora crassa (Dementhon et al. …

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