4.E.1 The Vacuolar (Acidocalcisome) Polyphosphate Polymerase (V-PPP) Family
Eukaryotes contain inorganic polyphosphate (polyP) and acidocalcisomes, which sequester polyP and store amino acids and divalent cations (Docampo and Moreno 2011; Docampo et al. 2013). Gerasimaitė et al. 2014 showed that polyP produced in the cytosol of yeast is toxic. Reconstitution of polyP translocation with purified vacuoles, the acidocalcisomes of yeast, showed that cytosolic polyP cannot be imported whereas polyP produced by the vacuolar transporter chaperone (VTC) complex, an endogenous vacuolar polyP polymerase, is efficiently imported and does not interfere with growth. PolyP synthesis and import require an electrochemical gradient, probably as a driving force for polyP translocation. VTC exposes its catalytic domain to the cytosol and has nine vacuolar TMSs. Mutations in the VTC transmembrane regions, which may constitute the translocation channel, block not only polyP translocation but also synthesis. Since they are far from the cytosolic catalytic domain of VTC, this suggests that the VTC complex obligatorily couples synthesis of polyP to its vesicular import in order to avoid toxic intermediates in the cytosol. The process therefore conforms to the classical definition of Group Translocation, where the substrate is modified during transport. Sequestration of otherwise toxic polyP may be one reason for the existence of this mechanism and acidocalcisomes (Gerasimaitė et al., 2014).
VTC2 has three recognized domains: an N-terminal SPX domain, a large central CYTH-like domain and an smaller transmembrane VTC1 (DUF202) domain. The SPX domain is found in Syg1, Pho81, XPR1 (SPX), and related proteins. This domain is found at the amino termini of a variety of proteins. In the yeast protein, Syg1, the N-terminus directly binds to the G-protein beta subunit and inhibits transduction of the mating pheromone signal. Similarly, the N-terminus of the human XPR1 protein binds directly to the beta subunit of the G-protein heterotrimer, leading to increased production of cAMP. Thus, this domain is involved in G-protein associated signal transduction. The N-termini of several proteins involved in the regulation of phosphate transport, including the putative phosphate level sensors, Pho81 from Saccharomyces cerevisiae and NUC-2 from Neurospora crassa, have this domain.
The SPX domains of the S. cerevisiae low-affinity phosphate transporters, Pho87 and Pho90, auto-regulate uptake and prevent efflux. This SPX-dependent inhibition is mediated by a physical interaction with Spl2. NUC-2 contains several ankyrin repeats. Several members of this family are annotated as XPR1 proteins: the xenotropic and polytropic retrovirus receptor confers susceptibility to infection with xenotropic and polytropic murine leukaemia viruses (MLV). Infection by these retroviruses can inhibit XPR1-mediated cAMP signaling and result in cell toxicity and death. The similarity between Syg1 phosphate regulators and XPR1 sequences has been noted, as has the additional similarity to several predicted proteins of unknown function, from Drosophila melanogaster, Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, S. cerevisiae, and many other diverse organisms.
CYTH-like superfamily enzymes hydrolyze triphosphate-containing substrates and require metal cations as cofactors. They have a unique active site located at the center of an eight-stranded antiparallel beta barrel tunnel (the triphosphate tunnel). The name CYTH originated from the gene designation for bacterial class IV adenylyl cyclases (CyaB), and from thiamine triphosphatase. Class IV adenylate cyclases catalyze the conversion of ATP to 3',5'-cyclic AMP (cAMP) and PPi. Thiamine triphosphatase is a soluble cytosolic enzyme which converts thiamine triphosphate to thiamine diphosphate. This domain superfamily also contains RNA triphosphatases, membrane-associated polyphosphate polymerases, tripolyphosphatases, nucleoside triphosphatases, nucleoside tetraphosphatases and other proteins with unknown functions.
The vacuolar transporter chaperone (VTC) complex integrates cytosolic polyP synthesis from ATP with polyP membrane translocation into the vacuolar lumen. In yeast and other eukaryotes, polyP synthesis is regulated by inositol pyrophosphate (PP-InsP) nutrient messengers, directly sensed by the VTC complex. Guan et al. 2023 reported the cryo-electron microscopy structure of signal-activated VTC complex at 3.0 Å resolution. Baker's yeast VTC subunits Vtc1, Vtc3, and Vtc4 assemble into a 3:1:1 complex. Fifteen trans-membrane helices form a novel membrane channel enabling the transport of newly synthesized polyP into the vacuolar lumen. PP-InsP binding orients the catalytic polymerase domain at the entrance of the trans-membrane channel, both activating the enzyme and coupling polyP synthesis to trans-membrane translocation. This provides mechanistic insights into the biogenesis of an ancient energy metabolite (Guan et al. 2023). Thus, the various proteins listed under different TC#s may be components of a single complex.
Liu et al. 2023 determined a cryo-EM structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure revealed a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. These findings, along with the functional data, suggests a mechanism of polyP channel gating and VTC complex activation (Liu et al. 2023).
The VTC complex also plays a role in vacuolar membrane fusion (Ogawa et al. 2000, Müller et al. 2003), and it is required for SEC18/NSF activity in SNARE priming, membrane binding of LMA1 and V0 trans-complex formation (Müller et al. 2002).
The generalized reaction catalyzed by polyphosphate polymerases is:
ATP + polyphosphate (P)n in the cytoplasm → ADP + polyphosphate (P)n+1 in the vacuolar lumen