SummaryRho GTPases have multiple, yet poorly defined functions during cytokinesis. By screening a Neurospora crassa knock-out collection for Rho guanine nucleotide exchange factor (GEF) mutants that phenocopy rho-4 defects (i.e. lack of septa, slow growth, abnormal branching and cytoplasmic leakage), we identified two strains defective in homologues of Bud3p and Rgf3 of budding and fission yeast respectively. The function of these proteins as RHO4-specific GEFs was determined by in vitro assays. In vivo microscopy suggested that the two GEFs and their target GTPase act as two independent modules during the selection of the septation site and the actual septation process. Furthermore, we determined that the N. crassa homologue of the anillinrelated protein BUD4 is required for septum initiation and that its deficiency leads to typical rho4 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.

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.

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.16370.001

Dipeptidyl peptidase 9 (DPP9) is a member of the S9B/DPPIV (DPP4) serine protease family, which cleaves N-terminal dipeptides at an Xaa-Pro consensus motif. Cytoplasmic DPP9 has roles in epidermal growth factor signalling and in antigen processing, whilst the role of the recently discovered nuclear form of DPP9 is unknown. Mice lacking DPP9 proteolytic activity die as neonates. We applied a modified 2D differential in-gel electrophoresis approach to identify novel DPP9 substrates, using mouse embryonic fibroblasts lacking endogenous DPP9 activity. A total of 111 potential new DPP9 substrates were identified, with nine proteins/peptides confirmed as DPP9 substrates by MALDI-TOF or immunoblotting. Moreover, we also identified the dipeptide Val-Ala as a consensus site for DPP9 cleavage that was not recognized by DPP8, suggesting different in vivo roles for these closely related enzymes. The relative kinetics for the cleavage of these nine candidate substrates by DPP9, DPP8 and DPP4 were determined. This is the first identification of DPP9 substrates from cells lacking endogenous DPP9 activity. These data greatly expand the potential roles of DPP9 and suggest different in vivo roles for DPP9 and DPP8.

SummaryCytokinesis is essential for cell proliferation, yet its molecular description is challenging, because > 100 conserved proteins must be spatially and temporally co-ordinated. Despite the high importance of a tight co-ordination of cytokinesis with chromosome and organelle segregation, the mechanism for determining the cell division plane is one of the least conserved aspects of cytokinesis in eukaryotic cells. Budding and fission yeast have developed fundamentally distinct mechanisms to ensure proper nuclear segregation. The extent to which these pathways are conserved in multicellular fungi remains unknown. Recent progress indicates common components, but different mechanisms that are required for proper selection of the septation site in the different groups of Ascomycota. Cortical cues are used in yeast-and filament-forming species of the Saccharomycotina clade that are established at the incipient bud site or the hyphal tip respectively. In contrast, septum formation in the filament-forming Pezizomycotina species Aspergillus nidulans and Neurospora crassa seems more closely related to the fission yeast programme in that they may combine mitotic signals with a cell end-based marker system and Rho GTPase signalling. Thus, significant differences in the use and connection of conserved signalling modules become apparent that reflect the phylogenetic relationship of the analysed models.

was previously considered as a purely cytosolic peptidase, localizes to the nucleus and is active there, raising the intriguing possibility that the longer DPP9 isoform may regulate the activity or stability of nuclear proteins, such as transcription factors.

No abstract

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 homologue 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 homolog 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. ZusammenfassungZytokinese 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 spezi...