Analyse der molekularen Funktion von Sterolen in der Membranfusion
Zusammenfassung der Projektergebnisse
The aim of the present study was to analyze the role of sterols in the process of membrane fusion. Although previous studies already showed that ergosterol is essential for the very first step of fusion of the yeast vacuole, called “priming”, recent results suggested another essential role in a downstream step of fusion. Using the routine assays of membrane tethering, trans-SNARE pairing, and lumenal compartment and lipid mixing established in my host lab, I analyzed in vitro membrane fusion in presence or absence of ergosterol. The results obtained in this study showed clearly that ergosterol is essential for membrane fusion in a system independent on the priming step. Therefore, ergosterol indeed has an additional function in the process. Importantly, both membrane tethering as well as trans-SNARE complex formation could be accomplished also without ergosterol present in the membrane. This suggested a requirement of ergosterol for lipid mixing, the final step of membrane fusion, probably by reducing the energy barrier. A structural feature of ergosterol is its small polar head group, similar to phosphatidylethanolamine or diacylglycerol. Therefore, the effect of replacing ergosterol by phosphatidylethanolamine instead of phosphatidylcholine was tested. Strikingly, proteoliposomes lacking sterols, but instead boring an increased amount of phosphatidylethanolamine, fused with an almost identical efficiency as proteoliposomes made up of complete vacuolar lipid mixture containing sterol. This again strongly suggests that sterols reduce the energy barrier of membrane fusion and thus are essential for fusion when physiological levels of each SNARE protein are present. In addition, these results indicate that it is possible to generate intermediates of fusion reactions. This is a prerequisite to analyze the order of the fusion pathway. Although, it was not possible to establish a system lacking ergosterol and adding it back to allow fusion at a distinct time point, another approach was successful. In the recently improved in vitro membrane fusion system of my host lab, Sec17 is virtually essential for fusion. It turned out that addition of Sec17 after a pre-incubation of the other components leads to a bust of fusion compared to a reaction having Sec17 from the beginning. This indicates the formation of a fusion intermediate waiting for Sec17. Since this effect can be observed in absence of Sec18, Sec17 apparently has an additional role in the final step of membrane fusion. The functional results obtained in this study which have been confirmed by physical interaction analysis and provide initial insights in the order of the fusion pathway from (i) HOPS tethering membranes by interaction with the Rab-protein Ypt7, (ii) formation of a HOPS:R:Qa:Qc trans-complex, (iii) addition of the Qb-SNARE to form a HOPS:4-SNARE trans-complex, (iv) binding of Sec17 and (v) lipid mixing to accomplish membrane fusion.
Projektbezogene Publikationen (Auswahl)
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(2017). Assembly of intermediates for rapid membrane fusion. J. Biol. Chem. pii: jbc.RA117.000791
Harner, M. and Wickner, W.
