Analysis of multiprotein-complex components pulled-down with STRIPAK in Sordaria macrospora
by Anika Groth
Date of Examination:2022-03-29
Date of issue:2022-04-14
Advisor:Prof. Dr. Stefanie Pöggeler
Referee:Prof. Dr. Stefanie Pöggeler
Referee:Prof. Dr. Heike Krebber
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Name:Anika Groth Dissertation 2022.pdf
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EnglishThe 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.
Keywords: STRIPAK; Sordaria macrospora; ARP1; dynein-dynactin complex; POM33; nuclear-pore complex; VAC14; Fab1p/PIKfyve-complex