Transport mechanisms of the putative vesicular nucleotide transporter VNUT

by Jennifer Struck
Doctoral thesis
Date of Examination:2024-10-22
Date of issue:2024-12-13
Advisor:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Reinhard Jahn
Referee:Prof. Dr. Silvio Rizzoli
Persistent Address: http://dx.doi.org/10.53846/goediss-10940

 

 

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Abstract

English

The vesicular nucleotide transporter VNUT (SLC17A9) is thought to refill recycled synaptic vesicles and other secretory vesicles with adenosine triphosphate (ATP) for chemical signal transmission. ATP translocation by VNUT has been proposed to rely on the electrical component (∆Ψ) of the electrochemical H+ gradient (ΔμH+) generated by the vacuolar-type H+ ATPase (Sawada et al., 2008). This PhD thesis focuses on characterising the poorly understood molecular transport mechanism(s) of VNUT in a minimal system. We successfully developed a fluorescence-based assay to measure real-time kinetics of ATP fluxes through VNUT reconstituted in liposomes. To generate a stable ΔμH+ across the liposomal membrane, the H+ pump TFoF1 from Bacillus thermophilus was co reconstituted with VNUT in the liposomes. Using this advanced in vitro system, no ΔμH+-dependent ATP influx through VNUT into the liposomal lumen was observed. Together with traditional radiolabelled ATP uptake assays, our results demonstrate that VNUT specifically binds but does not transport ATP. Key components, in addition to ΔμH+, that are potentially required for VNUT-dependent ATP translocation into the vesicle lumen are discussed. Using VNUT-TFoF1 liposomes and heterologous expression of VNUT in mammalian cells, we provide the first evidence that VNUT exhibits two transport modes for inorganic phosphate (Pi), similar to the closely related vesicular glutamate transporters VGLUTs (Cheret et al., 2021; Preobraschenski et al., 2018). When VNUT is localised in the vesicle membrane, VNUT mediates ΔμH+-dependent Pi transport into the vesicle lumen. Following exocytosis, VNUT is localised in the plasma membrane, where the transporter translocates Pi into the cytosol.
Keywords: synaptic vesicle; neurotransmitter; transporter; VNUT