Primary active proton transport by eukaryotic V-ATPases (vacuolar ATPases) is regulated via the reversible disassembly of the V1Vo holoenzyme into its peripheral catalytic V1 complex and its membrane-bound proton-translocating Vo complex. This nutrient-dependent phenomenon had been first detected in the midgut epithelium of non-feeding moulting tobacco hornworms (Manduca sexta) and in glucose-deprived yeast cells (Saccharomyces cerevisiae). Since reversible disassembly to date had been investigated mostly in vitro, we wanted to test this phenomenon under in vivo conditions. We used living yeast cells with V-ATPase subunits fused to green, yellow or cyan fluorescent protein and found that only the V1 subunit C (Vma5) was released into the cytosol after substitution of extracellular glucose with galactose, whereas the other V1 subunits remained at or near the membrane. FRET analysis demonstrated close proximity between V1 and Vo even under glucose-starvation conditions. Disassembly, but not reassembly, depended on functional microtubules. Results from overlay blots, pull-down assays and bimolecular fluorescence complementation support the assumption that subunit C interacts directly with microtubules without involvement of linker proteins.

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The (SLC45) was defined in the course of the Human Genome Project and consists of four members, A1-A4, which show only 20-30% identity of amino acid sequences among each other. All these members exhibit an identity of ∼20% to plant H/sucrose cotransporters. Recently, we expressed members of the murine SLC45 family in yeast cells and demonstrated that they are, like their plant counterparts, H/sucrose cotransporters. In contrast with the plant proteins, SLC45 transporters recognise also the monosaccharides glucose and fructose as physiological substrates and seem to be involved in alternative sugar supply as well as in osmoregulation of several mammalian tissues. In the present study, we provide novel insights into the regulation of SLC45 transporters. By screening for interaction partners, we found a 14-3-3 protein as a promising candidate for control of transport activity. Indeed, co-expression of the gamma isoform of murine 14-3-3 protein in yeast and oocytes led to a significant decrease in transport rates of the murine SLC45 transporters as well as of the plant H/sucrose transporter Sut1.