Catalytic Core of a Membrane-Associated Eukaryotic Polyphosphate Polymerase

Abstract

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Trypanosoma brucei Vacuolar Transporter Chaperone 4 (TbVtc4) Is an Acidocalcisome Polyphosphate Kinase Required for in Vivo Infection

“…The presence of PP i did not "prime" or stimulate TbVtc4 activity (see also Fig. 3C), as was observed with the yeast enzyme (13). On the other hand, the reported priming effect of PP i on the polyP kinase activity of ScVtc4p was confirmed by the increase in its efficiency in the presence of PP i with a k cat /K m ratio of 1.…”

“…E. coli (BL21 Codon Plus DE3 RIPL, Stratagene) were transformed with ScVtc4p(189 -480)/pQE-2. Histidinetagged ScVtc4p(189 -480) was induced overnight at 25°C with 0.5 M isopropyl-␤-D-thiogalactopyranoside, isolated by metal ion affinity chromatography, and desalted on a HiTrap column (GE Healthcare) with 25 mM Tris, 200 mM NaCl, 2 mM DTT (13).…”

“…A few genes encoding exopolyphosphatases (8 -11) and endopolyphosphatases (12) have been described in eukaryotes. Recently, the first eukaryotic enzyme involved in synthesis and translocation of polyP, ScVtc4p (Saccharomyces cerevisiae vacuolar transporter chaperone 4), was identified (13). The Vtc complex consists of four proteins (Vtc1-4) that form hetero-oligomeric complexes and are able to synthesize and transfer polyP into the vacuole as well as impacting membrane trafficking and vacuole fusion (14 -16).…”

Phosphorus sequestration in the form of polyphosphate by microbial symbionts in marine sponges

“…Polyphosphate kinase reversibly polymerizes phosphate to form high-energy bond polymers and is widespread in diverse prokaryote species. The gene responsible for the synthesis of polyP in eukaryotes was identified as the integral membrane vacuolar transporter chaperone complex (vtc4 gene) in yeast and diatoms (17). We searched the genome of the sponge Amphimedon queenslandica for potential ppk and vtc4 genes.…”

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

“…The yeast Saccharomyces cerevisiae is the only eukaryotic model where polyP physiology has been properly characterized by a genetic approach. In yeast, polyP synthesis is carried out by a subunit of the vacuolar transporter chaperone (VTC), Vtc4 (14).…”