Analysis of multiprotein-complex components pulled-down with STRIPAK in Sordaria macrospora

Der STRIPAK (striatin-interacting phosphatase and kinase)-Komplex ist in Pilzen und Tieren hoch konserviert. In dem filamentösen Ascomyceten Sordaria macrospora (Sm) kommt dem SmSTRIPAK eine wichtige Rolle bei der Hyphenfusion, sexuellen Entwicklung, Septierung und dem Wachstum zu. Durch Pulldown-Experimente mit SmSTRIPAK-Komponenten als Köder, konnten massenspektrometrisch eine Vielzahl möglicher Interaktionspartner identifiziert werden. Diese Arbeit gibt Einblicke in drei mögliche Interaktionspartner des SmSTRIPAK-Komplexes: ARP1 (actin-related protein 1), POM33 (pore-membrane protein of 33 kDa) und VAC14 (vacuolar-morphology protein 14). Sie alle sind selbst Komponenten großer und konservierter individueller Multiproteinkomplexe. ARP1 ist das am häufigsten vorkommende Protein in Dynactin und ist an dem retrograden Transport verschiedener Frachten auf Mikrotubuli durch den Dynein-Dynactin-Komplex beteiligt. In dieser Studie konnte gezeigt werden, dass SmARP1 für das Hyphenwachstum und die Pilzentwicklung wichtig ist. Mittels Fluoreszenzmikroskopie konnte gezeigt werden, dass die zelluläre Lokalisierung dieses Proteins dynamisch ist und es teilweise mit Zellkernen assoziiert ist, sowie subapikal in der Nähe des Spitzenkörpers (SPK) lokalisiert. Aufgrund dieser Charakteristika wurde SmARP1 als Markerprotein für aktiv wachsende Hyphen und Zellpolarität etabliert. Im zweiten Projekt wurde das Transmembranprotein POM33, welches in anderen Organismen mit dem Kernporenkomplex (NPC) assoziiert ist, analysiert. Dabei wurde gezeigt, dass die Eliminierung des Smpom33 Gens selbst unter Stressbedingungen keine Auswirkungen auf die sexuelle Entwicklung von S. macrospora hat. Darüber hinaus zeigten fluoreszenzmikroskopische Untersuchungen, dass SmPOM33 an der Kernhülle sowie am endoplasmatischen Retikulum (ER) lokalisiert. Nach einer massenspektrometrischen Analyse wurden Proteine des ERs als potenzielle Interaktions-partner identifiziert. Demnach ist SmPOM33 eher ein ER-Protein als ein Bestandteil des NPCs. Im dritten Projekt dieser Arbeit wurde das Protein VAC14 untersucht. Es ist die gerüstbildende Untereinheit des Fab1p/PIKfyve-Komplexes, der in Hefe an der Vakuolenmembran und in Säugetieren an den Membranen von Endolysosomen lokalisiert ist. Er regelt den Umsatz und die Synthese von PtdIns(3,5)P2, welches bei zahlreichen zellulären Prozessen wie Organellenmorphologie, Ansäuerung von Endolysosomen und Autophagie beteiligt ist. In dieser Studie wurde VAC14 zum ersten Mal in filamentösen Pilzen untersucht. Demnach kolokalisiert SmVAC14 mit dem ER, Golgi, vesikulären Membranen, frühen und späten Endosomen und der SmSTRIPAK-Komponente SCI1. Darüber hinaus zeigte die ∆vac14 Mutante deformierte Perithezien, eine Beeinträchtigung der Ascosporenbildung sowie eine veränderte Vakuolenmorphologie. Des Weiteren ist eine ∆vac14 Mutante sensitiver gegenüber verschiedenen Arten von Stress, ist jedoch nicht in dem Prozess der Autophagie beeinträchtigt. Diese Arbeit zeigt, dass die drei hier analysierten Proteine selbst Teil separater Multiproteinkomplexe sind und dass sowohl ARP1 als auch VAC14 für die korrekte sexuelle Entwicklung in S. macrospora erforderlich sind.The striatin-interacting phosphatase and kinase (STRIPAK)-complex is highly conserved and can be found in fungi and animals. In the filamentous ascomycete Sordaria macrospora (Sm), the SmSTRIPAK plays an important role in hyphal fusion, sexual development, septation and growth. In previous studies, pulldown experiments of SmSTRIPAK-components as baits coupled to liquid chromatography-mass spectrometry (LC-MS) provided a variety of possible interaction partners. This work gives insights into three of these pulled-down proteins, namely: the actin-related protein 1 (ARP1), the pore-membrane protein of 33 kDa (POM33) and the vacuolar-morphology protein 14 (VAC14), which are all part of large and conserved individual multiprotein complexes themselves. ARP1 is the most abundant protein in dynactin and is involved in mediating retrograde transport of various cargos on microtubules via the dynein-dynactin complex. In this study, SmARP1 was shown to be important for proper hyphal growth and fungal development. Fluorescence microscopy revealed that its localization is a dynamic process and that SmARP1 localizes subapical to the Spitzenkörper (SPK) as well as in close association with nuclei. Due to these characteristics, SmARP1 was established as a marker protein for actively growing hyphae and cell polarity. In the second project, the transmembrane protein POM33 as putative part of the nuclear-pore complex (NPC) was analyzed. Thereby it was shown that deletion of Smpom33 has no impact on sexual development even under stress conditions. Additionally, fluorescence microscopic investigations showed that SmPOM33 localizes at the nuclear envelope (NE) and the endoplasmic reticulum (ER). After LC-MS analysis, proteins of the ER were identified as potential interactors. This led to the conclusion that SmPOM33 is rather an ER protein than a component of the NPC. In the third project, VAC14 as scaffolding subunit of the Fab1p/PIKfyve-complex was investigated. Vac14p/ArPIKfyve localizes to the vacuolar membrane in yeast and to membranes of endolysosomes in mammals, respectively. There, it facilitates the turnover and synthesis of PtdIns(3,5)P2 that functions in multiple processes including organelle morphology, acidification of endolysosomes and autophagy. The data presented here investigated VAC14 for the first time in a filamentous fungus. In S. macrospora, SmVAC14 co-localizes with the ER, Golgi, vacuolar membranes, early and late endosomes and the SmSTRIPAK-component SCI1. Moreover, the ∆vac14 mutant showed deformed perithecia, impairment of ascospore formation, and altered vacuolar morphology. Furthermore, the ∆vac14 mutant displayed an increased sensitivity to diverse types of stresses; however, autophagy was not affected. This work revealed that the three proteins presumably interacting with components of the STRIPAK-complex analyzed here, likewise are part of separate multiprotein complexes, and ARP1 and VAC14 are also required for accurate sexual development in S. macrospora.2023-03-2

The vacuolar morphology protein VAC14 plays an important role in sexual development in the filamentous ascomycete Sordaria macrospora

Abstract The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P 2 ) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P 2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located to vacuolar membranes in yeast and to endosomal membranes in mammals, where it controls the synthesis and turnover of PtdIns(3,5)P 2 . In this study, we analyzed the role and function of the Fab1p/PIKfyve-complex scaffold protein SmVAC14 in the filamentous ascomycete Sordaria macrospora (Sm). We generated the Smvac14 deletion strain ∆vac14 and performed phenotypic analysis of the mutant. Furthermore, we conducted fluorescence microscopic localization studies of fluorescently labeled SmVAC14 with vacuolar and late endosomal marker proteins. Our results revealed that SmVAC14 is important for maintaining vacuolar size and appearance as well as proper sexual development in S. macrospora . In addition, SmVAC14 plays an important role in starvation stress response. Accordingly, our results propose that the turnover of PtdIns(3,5)P 2 is of great significance for developmental processes in filamentous fungi.Deutsche Forschungsgemeinschaft http://dx.doi.org/10.13039/501100001659Georg-August-Universität Göttingen http://dx.doi.org/10.13039/50110000338

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life

Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora

In the filamentous fungus Sordaria macrospora (Sm), the STRIPAK complex is required for vegetative growth, fruiting-body development and hyphal fusion. The SmSTRIPAK core consists of the striatin homolog PRO11, the scaffolding subunit of phosphatase PP2A, SmPP2AA, and its catalytic subunit SmPP2Ac1. Among other STRIPAK proteins, the recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Pulldown experiments with SCI identified the transmembrane nucleoporin (TM Nup) SmPOM33 as a potential nuclear-anchor of SmSTRIPAK. Localization studies revealed that SmPOM33 partially localizes to the nuclear envelope (NE), but mainly to the endoplasmic reticulum (ER). We succeeded to generate a Δpom33 deletion mutant by homologous recombination in a new S. macrospora Δku80 recipient strain, which is defective in non-homologous end joining. Deletion of Smpom33 did neither impair vegetative growth nor sexual development. In pulldown experiments of SmPOM33 followed by LC/MS analysis, ER-membrane proteins involved in ER morphology, protein translocation, glycosylation, sterol biosynthesis and Ca2+-transport were significantly enriched. Data are available via ProteomeXchange with identifier PXD026253. Although no SmSTRIPAK components were identified as putative interaction partners, it cannot be excluded that SmPOM33 is involved in temporarily anchoring the SmSTRIPAK to the NE or other sites in the cell