Biophysical Society Conference | Estes Park 2023

Membrane Budding and Fusion

Monday Speaker Abstracts

RECONSTRUCTING THE COLLECTIVE BEHAVIOUR OF THE ENDOSOMAL MEMBRANE FUSION MACHINERY Lisa Redlingshöfer 1,2 ; Mareike Jordan 1 ; Swantje Lenz 1 ; Alexander von Appen 1 ; Stephan Grill 1,2 ; Marino Zerial 1 ; 1 Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany 2 Technische Universität Dresden, Dresden, Germany The Rab5 GTPase determines the location and timing of endosomal membrane fusion. In order to do so, Rab5 is activated on early endosomal membranes by the Rabex5:Rabaptin5 complex, which enables the recruitment of a plethora of Rab5 effector proteins and the formation of high molecular weight oligomers. Yet, in the absence of PI3P, the hallmark phosphoinositide lipid of the early endosome, Rab5 is unable to recruit the full fusion machinery. To understand how the interplay of protein-protein and protein-lipid interactions creates a fully functional collective, we probed the assembly dynamics and characterised the structural arrangement of the endosomal fusion machinery. To this end, we combined in vitro reconstitution experiments on liposomes and supported lipid bilayers with cryoEM, crosslinking mass spectrometry and TIRF microscopy analysis. Our investigations revealed a novel binding interaction of Rabex5:Rabaptin5 with PI3P, which could serve to target Rab5 to early endosomes. Varying the concentration of PI3P or combination of effector proteins affected assembly dynamics and morphology. Furthermore, we found that Rabex5:Rabaptin5 displays significant conformational variability, which is likely to affect its ability to bind to PI3P and other Rab5 effector proteins. Our findings suggest that PI3P assumes a critical role in mediating the protein-protein interactions necessary to assemble the full Rab5-mediated membrane fusion machinery.

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