The Functional Role of the Membrane Bending Ability of the Yeast PROPPINs

by Jan Dennis Förster
Doctoral thesis
Date of Examination:2025-01-30
Date of issue:2025-04-04
Advisor:Prof. Dr. Michael Thumm
Referee:Dr. Ricarda Richter-Dennerlein
Referee:Prof. Dr. Stefan Jakobs
Referee:PD Dr. Antje Ebert
Referee:Dr. Alex Faesen
Referee:Prof. Dr. Elisa Oberbeckmann
Persistent Address: http://dx.doi.org/10.53846/goediss-11187

 

 

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Abstract

English

The meticulously regulated mechanism, which overseas processes of cellular breakdown and recycling, is called macroautophagy. It has been preserved from yeast to more advanced eukaryotic organisms. As a response to various external and internal stimuli it operates continuously at a low baseline and degrades parts of the cytosol selectively as well as non-selectively. It is initiated with the de novo creation of the so- called isolation membrane or phagophore, a cup-shaped membrane structure, emerging at the pre-autophagosomal structure (PAS). The phagophore expands to subsequently form a closed, double-membrane structure called the autophagosome. Cellular cargo situated at the growing phagophore is completely encased before the structure is sealed off. The autophagosome eventually fuses with the vacuole to release its contents into the vacuolar lumen for degradation and recycling. The formation of the autophagosome necessitates the production of phosphatidylinositol 3-phophate (PI3P) at the PAS. This facilitates the attachment of b-propellers that bind polyphosphoinositides (PROPPINs), which are a well-conserved group of WD40-repeat proteins that typically adopt a seven-bladed b-propeller structure. PROPPINs moderate protein-protein interactions, such as scaffolding or assembly and regulation of multi-subunit complexes, due to their WD40 domain. Additionally, PROPPINs feature a FRRG motif, which is highly conserved, at their fifth and sixth propeller blade. This motif enables the binding of phosphoinositides, namely two molecules of PI3P or PI(3,5)P2. Three PROPPINs have been identified in Saccharomyces cerevisiae: Atg18, Atg21 and Hsv2. While they exhibit high homology, each fulfills distinct functions during different subtypes of autophagy. Atg18 is essential for all forms of autophagy depending on PI3P, which makes it a core autophagy protein. Furthermore, Atg18 contributes to non- autophagic functions at the vacuole, which are related to vacuolar morphology and fragmentation. In contrast, Atg21 is not essential for non-selective autophagy but carries out vital functions during selective autophagic processes, such as the Cvt pathway, which specializes in transport of vacuolar hydrolases like prApe1 to the vacuole. Opposite of these well-defined PROPPINs stands Hsv2, which is the least characterized of the three. It has small implications for piecemeal microautophagy of the nucleus, which marks the only known detection impacting autophagy. This study focused on investigating the membrane bending ability of the yeast PROPPINs. This membrane bending is driven by the partial insertion of an amphipathic a-helix, which is formed by the loop 6CD of the PROPPINs upon contact with the bilayer. Since Hsv2 is the least characterized PROPPIN, this study put most emphasize on it and its potential autophagic and non-autophagic functions. Mutants of all three PROPPINS were designed and tested, which lack the ability to form an intact amphipathic a-helix and induce membrane curvature and fission. The first experiments conducted in this study addressed the relevance of the membrane bending ability for the autophagic functions of Atg18 and Atg21. The autophagic assays showed that Atg21 needs its membrane bending ability for prApe1 maturation during the Cvt pathway and also to a lesser extent during bulk autophagy under starvation conditions. Atg18 and Hsv2 showed no involvement of their membrane bending ability during their autophagic functions. Nonetheless, we could show that the autophagy of large cargos depends on Hsv2. Here, Hsv2 binds to the growing edges of the phagophore and supports Atg18 in its function to facilitate growth of the isolation membrane. Previous studies in our lab showed that Atg18 is able to bind to the retromer complex and regulate vacuolar morphology and vacuolar fragmentation. My experiments show, that Hsv2 is also able to bind to the retromer complex, especially in absence of Atg18 and mediate vacuolar fragmentation. Moreover, I could show that the membrane bending ability of Hsv2 is necessary for its retromer function. In addition, deletion of Hsv2 led to mislocalization of the t-SNARE Pep12 in diploid yeast cells. This indicates an additional function of Hsv2 with retromer that involves recycling of proteins. Since we found a function of Hsv2, restricted to diploid cells, we assumed an involvement of Hsv2 during sporulation, which was already hinted to in other studies. My sporulation experiments showed that hsv2∆ cells exhibit an impaired spore wall phenotype. Furthermore, we determined that Gsc2 and Chs3, two proteins involved in spore wall assembly, are mislocalizing in deletion cells as well as in mutated Hsv2 cells that no longer can form an intact a-helix in loop 6CD and induce membrane bending. This led to the conclusion, that Hsv2 retromer complex is involved in recycling of proteins during sporulation.
Keywords: Saccharomyces cerevisiae; Autophagy; Atg18; Atg21; Retromer; Sporulation; 6CD loop
Schlagwörter: Saccharomyces cerevisiae; Autophagy; Hsv2; Atg18; Atg21; Retromer; Sporulation; 6CD loop