4 research outputs found
Regulierung der Septenbildung in Neurospora crassa durch die RHO4 GTPase
Zytokinese bezeichnet den Prozess, bei dem eine einzelne Zelle in zwei Tochterzellen geteilt wird, und ist bei Eukaryoten hoch konserviert. Die Rho GTPasen sind in ihrer Funktion als Schlüsselregulatoren zahlreicher zellulärer Prozesse auch wesentlich an der Zellteilung beteiligt. Über die genauen Funktionen der Rho Proteine und deren Regulation in diesem Prozess war zu Beginn meiner Arbeit jedoch wenig bekannt. Die Rho GTPase RHO4 des Euascomyceten Neurospora crassa ist essentiell für die Septierung. Im Rahmen dieser Arbeit wurde die N. crassa Knockout-Stammsammlung nach Deletionsmutanten durchsucht, die dieselben Defekte zeigten wie die rho-4 Mutante, d.h. fehlende Septen, dünne Hyphen, abnormes Verzweigungsmuster, langsames Wachstum und zytoplasmatisches Auslaufen. Hierbei wurden die zwei Guaninnukleotid-Austauschfaktoren (GEFs) BUD3 und RGF3 identifiziert, die nach ihren Homologen Bud3p und Rgf3p in Saccharomyces cerevisiae und Schizosaccharomyces pombe benannt wurden. Mittels in vitro GEF-Assays wurde gezeigt, dass beide Proteine spezifisch die GDP/GTP-Austauschaktivität von RHO4 stimulieren. Auf gleiche Weise wurde das in Aspergillus nidulans vorkommende BUD3 Homolog AnBud3 als in vitro GDP/GTP-Austauschfaktor von A. nidulans AnRho4 und N. crassa RHO4 identifiziert. Diese Ergebnisse deuten daraufhin, dass es sich bei dem RHO4-BUD3 Modul um ein konserviertes Modul innerhalb der Euascomyceten handeln könnte. In vivo Lokalisationsstudien zeigten, dass BUD3 und RGF3 wie die Rho GTPase RHO4 an Septen akkumulieren. Anschließende Untersuchungen zu Lokalisationsabhängigkeiten lassen vermuten, dass die beiden GEFs jeweils zusammen mit RHO4 als zwei voneinander unabhängige Module in der Septierung agieren. Das Modul BUD3-RHO4 scheint hierbei eher an der Festlegung der Septierungsstelle beteiligt zu sein, während RGF3-RHO4 seine Funktion womöglich in dem eigentlichen Septierungsprozess ausübt. Neben den beiden GEFs wurde das Anillin-ähnliche Protein BUD4 als weiteres essentielles Protein für die Septierung in N. crassa identifiziert. Die bud-4 Deletionsmutante wies die oben beschriebenen rho-4 Defekte auf. BUD4 akkumulierte wie BUD3, RGF3 und RHO4 an Septen in vivo. Seine Lokalisation als kortikaler Ring an der zukünftigen Septierungsstelle, noch vor der eigentlichen Septenbildung, war unabhängig von funktionellem BUD3 oder RGF3. Dies legt den Schluss nahe, dass BUD4 oberhalb von beiden RHO4-Modulen als kortikales Markerprotein für die Septierungsstelle agiert. Da zudem BUD3 und BUD4 sowie RHO4 an der Pore vollständig ausgebildeter Septen verblieben, ist es möglich, dass diese Proteine dort zusätzliche Funktionen besitzen.Cytokinesis, the process in which a single cell is separated into two daughter cells, is highly conserved from plants to fungi and animals. Consistent with being key regulators of many cellular processes Rho GTPases have multiple, yet poorly-defined functions during cytokinesis. The Rho GTPase RHO4 in the euascomycete Neurospora crassa is essential for septation. By screening a N. crassa knockout collection for deletion mutants that phenocopy rho-4 defects (i.e. lack of septa, thin hyphae, slow growth, abnormal branching and cytoplasmic leakage), two strains defective in putative Rho guanine nucleotide exchange factors (GEFs) were identified. They were named BUD3 and RGF3 according to their homologues Bud3p and Rgf3p in budding and fission yeast, respectively. The function of these two proteins as RHO4-specific GEFs was determined by in vitro GEF assays. Likewise, AnBud3, the BUD3 homolog in Aspergillus nidulans, was identified as GEF of A. nidulans AnRho4 and N. crassa RHO4 in vitro, indicating that the Rho4-Bud3 module and its function during septation is conserved throughout the euascomycete lineage. In vivo microscopy with GFP fusion proteins suggested that the two N. crassa GEFs and their target GTPase RHO4 function as two independent modules during septation with BUD3-RHO4 acting in site selection and RGF3-RHO4 acting in the actual septation process. Furthermore, the N. crassa homologue of the anillin-related protein BUD4 was determined as being required for septum initiation. Its deficiency leads to typical rho-4 defects. Localization of BUD4 as a cortical ring prior to septation initiation was independent of functional BUD3 or RGF3. These data position BUD4 upstream of both RHO4 functions in the septation process and make BUD4 a prime candidate for a cortical marker protein involved in the selection of future septation sites. The persistence of both BUD proteins and of RHO4 at the septal pore suggests additional functions of these proteins at mature septa
Regulation of Septum Formation by the Bud3–Rho4 GTPase Module in \u3ci\u3eAspergillus nidulans\u3c/i\u3e
The ability of fungi to generate polarized cells with a variety of shapes likely reflects precise temporal and spatial control over the formation of polarity axes. The bud site selection system of Saccharomyces cerevisiae represents the best-understood example of such a morphogenetic regulatory system. However, the extent to which this system is conserved in the highly polarized filamentous fungi remains unknown. Here, we describe the functional characterization and localization of the Aspergillus nidulans homolog of the axial bud site marker Bud3. Our results show that AnBud3 is not required for polarized hyphal growth per se, but is involved in septum formation. In particular, our genetic and biochemical evidence implicates AnBud3 as a guanine nucleotide exchange factor for the GTPase Rho4. Additional results suggest that the AnBud3–Rho4 module acts downstream of the septation initiation network to mediate recruitment of the formin SepA to the site of contractile actin ring assembly. Our observations provide new insight into the signaling pathways that regulate septum formation in filamentous fungi
Regulation of Septum Formation by the Bud3–Rho4 GTPase Module in Aspergillus nidulans
The ability of fungi to generate polarized cells with a variety of shapes likely reflects precise temporal and spatial control over the formation of polarity axes. The bud site selection system of Saccharomyces cerevisiae represents the best-understood example of such a morphogenetic regulatory system. However, the extent to which this system is conserved in the highly polarized filamentous fungi remains unknown. Here, we describe the functional characterization and localization of the Aspergillus nidulans homolog of the axial bud site marker Bud3. Our results show that AnBud3 is not required for polarized hyphal growth per se, but is involved in septum formation. In particular, our genetic and biochemical evidence implicates AnBud3 as a guanine nucleotide exchange factor for the GTPase Rho4. Additional results suggest that the AnBud3–Rho4 module acts downstream of the septation initiation network to mediate recruitment of the formin SepA to the site of contractile actin ring assembly. Our observations provide new insight into the signaling pathways that regulate septum formation in filamentous fungi
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DPP9 is a novel component of the N-end rule pathway targeting the tyrosine kinase Syk
The aminopeptidase DPP9 removes dipeptides from N-termini of substrates having a proline or alanine in second position. Although linked to several pathways including cell survival and metabolism, the molecular mechanisms underlying these outcomes are poorly understood. We identified a novel interaction of DPP9 with Filamin A, which recruits DPP9 to Syk, a central kinase in B-cell signalling. Syk signalling can be terminated by degradation, requiring the ubiquitin E3 ligase Cbl. We show that DPP9 cleaves Syk to produce a neo N-terminus with serine in position 1. Pulse-chases combined with mutagenesis studies reveal that Ser1 strongly influences Syk stability. Furthermore, DPP9 silencing reduces Cbl interaction with Syk, suggesting that DPP9 processing is a prerequisite for Syk ubiquitination. Consistently, DPP9 inhibition stabilizes Syk, thereby modulating Syk signalling. Taken together, we demonstrate DPP9 as a negative regulator of Syk and conclude that DPP9 is a novel integral aminopeptidase of the N-end rule pathway. DOI: http://dx.doi.org/10.7554/eLife.16370.00