Lipid transporters are integral membrane proteins that play a critical role in supporting essential cellular processes and are crucial for the virulence of the pathogenic yeast Cryptococcus neoformans. Cryo-electron microscopy has recently resolved the structures of several ATP-dependent lipid transporters, but functional characterization remains a major challenge. Current concepts suggest that the activity of lipid transporters is regulated by lipid bilayer composition, membrane curvature, and co-factors. However, direct experimental evidence for these concepts is lacking. Not only is the purification of active integral membrane proteins limited, but sophisticated techniques are required to study membrane protein activity at the molecular level. Our approach will tackle this problem from both sides by establishing defined reconstituted vesicle systems based on large unilamellar liposomes and state-of-the-art single-molecule techniques to study membrane transporters. We have developed a fluorescence-based microscopy setup to analyse single vesicles, allowing us to extract information hidden in traditional ensemble-averaged biophysical or biochemical studies that are hampered by sample heterogeneity. This high-throughput approach allows the parallel analysis of multiple parameters of individual proteoliposomes, including size and protein copy number, with the aim of correlating these features with protein activity. We have recently established the setup and provide now a proof-of-principle for monitoring the scramblase activity of reconstituted VDAC1 dimers at the single vesicle level.

Sarina Veit¹, Anant K. Menon², Thomas Günther Pomorski^1,3

¹Department of Molecular Biochemistry, Faculty of Chemistry and Biochemistry, Ruhr University Bochum, Bochum 44801, Germany

²Department of Biochemistry, Weill Cornell Medical College, New York, New York 10021, USA

³Department of Plant and Environmental Sciences, University of Copenhagen, DK-1871 Frederiksberg C, Denmark