The a-Subunit of the V-type H+-ATPase Interacts with Phosphofructokinase-1 in Humans

Abstract

V-type or H؉ -ATPases are a family of ATP-dependent proton pumps that move protons across the plasma membrane at specialized sites such as kidney epithelial cells and osteoclasts as well as acidifying intracellular compartments. The 100-kDa polytopic a-subunit of this group of ATPases is suggested to play an important role in coupling the two functions of the pump, ATP hydrolysis and proton transport. In man, different a-subunit isoforms are encoded by four genes. ATP6V0A4 encodes a4, which is expressed apically in ␣-intercalated cells in both human and mouse kidney. We sought binding partners for the C terminus of a4 in order to address its potential role in the H ؉ -ATPase complex. Random peptide phage display analysis revealed a consensus motif (WLELRP) with almost complete homology to part of the enzyme phosphofructokinase 1 (PFK-1). Activity of this enzyme is the rate-limiting step in glycolysis. Specificity of a4 binding to this peptide was confirmed by enzymelinked immunosorbent assay. Protein-protein interaction was further demonstrated by co-immunoprecipitation of a4 with PFK-1 from solubilized human kidney membrane proteins. An in vitro bead-bound PFK-1 pulldown assay showed that this interaction was also true for the ubiquitously expressed a1 subunit. Finally, PFK-1 co-immunolocalized with a4 in ␣-intercalated cells in the collecting ducts of human kidney. These findings indicate a direct link between V-type H ؉ -ATPases and glycolysis via the C-terminal region of the a-subunit of the pump and suggest a novel regulatory mechanism between H ؉ -ATPase function and energy supply. This interaction between the a-subunit and PFK-1 also provides new evidence that the C terminus of this subunit lies cytoplasmically in vivo.The H ϩ -, vacuolar-type or V-ATPases (hereafter abbreviated H ϩ -ATPases) are a family of ATP-dependent proton pumps that acidify intracellular compartments in all cells. They also pump protons across the plasma membranes of osteoclasts and of certain epithelial cells in the kidney and male genital tract. H ϩ -ATPases from fungi, plants, and animals are structurally very similar to each other (1), consisting of two major functional domains known as V 1 and V 0 . The V 1 domain is composed of at least eight different subunits (A-H) and contains ATP binding sites involving the A and B subunits. This domain is mainly responsible for ATP hydrolysis. The V 0 domain contains up to five additional subunits (a, c, cЈ, cЉ, and d) and is mainly responsible for proton translocation across the membrane in which it is anchored (2).

Analysis of the Membrane Topology of Transmembrane Segments in the C-terminal Hydrophobic Domain of the Yeast Vacuolar ATPase Subunit a (Vph1p) by Chemical Modification

“…4a), indicating a cytoplasmic orientation. A cytoplasmic orientation of the C-terminal tail is also in agreement with previous studies showing that the C terminus of subunit a is able to bind the glycolytic enzyme phosphofructokinase, as demonstrated by co-immunoprecipitation (24), and that a GFP tag attached at the C terminus of subunit a from Dictyostelium can be proteolytically removed in intact endosomes (25). The former result does not rule out an extracellular orientation for the C terminus of subunit a as a number of cytoplasmic proteins (such as HSP90) have been shown to be released into the extracellular medium (45).…”

“…Previous data from our laboratory analyzing the membrane topology of subunit a led to a model of the protein in which the cytoplasmic N-terminal domain was followed by a C-terminal domain containing nine transmembrane helices, placing the C terminus on the lumenal side of the membrane (15). Other data, however, have suggested that the C terminus is located on the cytoplasmic rather than the lumenal side of the membrane (24,25). To further address the membrane topology of subunit a, we have employed a combination of cysteine-scanning mutagenesis and chemical modification with membrane permeant and impermeant reagents.…”

“…Starting with this Cys-less form of Vph1p, thirty single cysteine-containing mutants were constructed by site-directed mutagenesis. Because the topology of the C-terminal most portion of Vph1p was in dispute (15,24,25), and because all of the residues shown to be important for proton transport had been localized to this region (12,19,20), we focused our mutagenesis beginning at residue Gly-602 in the putative loop between TM5 and TM6 and continuing to the C-terminal residue Ser-840. Each of the single cysteine containing mutants of Vph1p was expressed in MM112 using the single copy plasmid pRS316 and the growth phenotype of the cells analyzed at pH 7.5.…”

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…Crosslinking, two-hybrid screening, coimmunoprecipitation, and electron microscopy showed that the a-subunit isoforms directly interact with A-, E-, H-, C-, and G-subunits of the V 1 sector [9][10][11][12][13] as well as with c-and d-subunits of the V o sector. 2,[14][15][16] Recently, it has also been shown that the V-ATPase a-subunit directly assembles with proteins that are not part of the V-ATPase complex including: (i) aldolase B, 17,18 (ii) phosphofrucktokinase-1, 19,20 (iii) calmodulin, 21 and (iv) t-SNARES syntaxin and SNAP-25. 22 However, the mechanisms and molecular details of interactions with these proteins remain poorly understood, mainly due to the lack of a high-resolution structure of the a-subunit and the absence of studies designed for mapping interaction sites on both the a-subunit and its associated proteins.…”

Saccharomyces cerevisiae Lacking Btn1p Modulate Vacuolar ATPase Activity to Regulate pH Imbalance in the Vacuole

“…During the logarithmic phase of growth, high glucose levels of the medium lead to the synthesis of high amounts of ATP by the glycolytic pathway. Several studies have demonstrated a direct interaction between glycolytic enzymes, such as aldolase (43)(44)(45) or phosphofructokinase-1 (46), and several subunits of the V-ATPase. Moreover, it has been postulated that the V-ATPase and glycolytic enzymes form a complex in the vacuolar membrane to maximize the efficiency of energy provision to acidify the vacuole.…”