RESUMEN
Aspergillus nidulans gapA1, a mutation leading to compact, fluffy colonies and delayed polarity establishment, maps to a gene encoding a Ras GTPase-activating protein. Domain organization and phylogenetic analyses strongly indicate that GapA regulates one or more "true" Ras proteins. A gapADelta strain is viable. gapA colonies are more compact than gapA1 colonies and show reduced conidiation. gapADelta strains have abnormal conidiophores, characterized by the absence of one of the two layers of sterigmata seen in the wild type. gapA transcript levels are very low in conidia but increase during germination and reach their maximum at a time coincident with germ tube emergence. Elevated levels persist in hyphae. In germinating conidiospores, gapADelta disrupts the normal coupling of isotropic growth, polarity establishment, and mitosis, resulting in a highly heterogeneous cell population, including malformed germlings and a class of giant cells with no germ tubes and a multitude of nuclei. Unlike wild-type conidia, gapADelta conidia germinate without a carbon source. Giant multinucleated spores and carbon source-independent germination have been reported in strains carrying a rasA dominant active allele, indicating that GapA downregulates RasA. gapADelta cells show a polarity maintenance defect characterized by apical swelling and subapical branching. The strongly polarized wild-type F-actin distribution is lost in gapADelta cells. As GapA-green fluorescent protein shows cortical localization with strong predominance at the hyphal tips, we propose that GapA-mediated downregulation of Ras signaling at the plasma membrane of these tips is involved in the polarization of the actin cytoskeleton that is required for hyphal growth and, possibly, for asexual morphogenesis.
Asunto(s)
Actinas/metabolismo , Aspergillus nidulans/crecimiento & desarrollo , Polaridad Celular , Citoesqueleto , Hifa/fisiología , Esporas Fúngicas/crecimiento & desarrollo , Proteínas Activadoras de ras GTPasa/metabolismo , Secuencia de Aminoácidos , Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Membrana Celular/metabolismo , Clonación Molecular , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Filogenia , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Esporas Fúngicas/genética , Proteínas Activadoras de ras GTPasa/genéticaRESUMEN
The Aspergillus nidulans UapC protein is a high-affinity, moderate-capacity, uric acid-xanthine transporter, which also displays a low transport capacity for hypoxanthine, adenine, and guanine. It has been previously shown that a functional UapC-GFP fusion protein localises at the plasma membrane. Here, we demonstrate that ammonium, a preferred nitrogen source, dramatically changes the subcellular distribution of UapC. After addition of ammonium, UapC-GFP is removed from the plasma membrane and is concentrated into the vacuolar compartment. A chimeric gene construct in which an inducible promoter, insensitive to nitrogen repression, drives the expression of UapC-GFP, allowed us to demonstrate that the ammonium-dependent redistribution of UapC can be dissociated from the transcriptional repression of the gene. These results provide further support for the occurrence of endocytosis and the lysosomal-endosomal function of the vacuolar compartment in A. nidulans.