Reversible disassembly of the yeast V-ATPase revisited under in vivo conditions

Abstract

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.

The vacuolar-type H+-ATPase at a glance – more than a proton pump

“…Aldolase interacts with the VATPase subunits a, B and E, and this interaction is required to stabilize the assembled V-ATPase on the membrane (Lu et al, 2007;Lu et al, 2001;Lu et al, 2004;Merkulova et al, 2010;Merkulova et al, 2011). Indeed, aldolase separates from the V-ATPase upon glucose starvation , underlying the reported dissociation of the V 1 and V 0 domains in a microtubuledependent manner (Tabke et al, 2014;Xu and Forgac, 2001). Interestingly, although it does stabilize the V 1 -V 0 complex, aldolase is not required for the reassembly of the dissociated domains when glucose is reintroduced; the regulator of the H + -ATPase of vacuolar and endosomal membranes (RAVE) complex (comprising Skp1, Rav1 and Rav2) is responsible for the reassembly process, which also requires phosphatidylinositol 3-kinase (Seol et al, 2001;Smardon et al, 2002).…”

“…In the latter system the dissociation is dependent on an interaction between the V-ATPase with microtubules (Xu and Forgac, 2001). Recent evidence suggests that the subunit C of the V 1 domain (see Box 1) directly interacts with microtubules and is the sole component that dissociates during glucose withdrawal (Tabke et al, 2014). The mechanisms underlying the dissociation and its implications as a possible metabolic sensor are discussed below in the section describing non-canonical functions of the V-ATPase.…”

Molecular Interactions and Cellular Itinerary of the Yeast RAVE (Regulator of the H+-ATPase of Vacuolar and Endosomal Membranes) Complex

“…This subunit binds at the interface of V 1 and V o and is released from both sectors upon glucose deprivation (6,14,17). The two-hybrid experiments in Fig.…”

“…The V 1 C subunit is present at the interface of the V 1 and V o sectors and is thus positioned to play a critical role in reversible disas-sembly of the V-ATPase (14 -16). Interestingly, this subunit is released from both the V 1 and V o sectors when the V-ATPase disassembles (6,17). Subunit C binds to Rav1 independently of the V 1 subcomplex and is the only subunit that has shown any binding to Rav2 (18).…”

Regulation of Vacuolar H+-ATPase (V-ATPase) Reassembly by Glycolysis Flow in 6-Phosphofructo-1-kinase (PFK-1)-deficient Yeast Cells

“…Upon glucose depletion, V 1 subunit C is released into the cytoplasm, causing the rest of the V 1 domain to dissociate from V o (10,17,18). Disassembly completely inactivates V-ATPase pumps because V 1 without V o cannot hydrolyze ATP, and V o without V 1 cannot transport protons (2).…”