Characterization of vacuolar amino acid transporter from <i>Fusarium oxysporum</i> in <i>Saccharomyces cerevisiae</i>

<p><i>Fusarium oxysporum</i> causes wilt disease in many plant families, and many genes are involved in its development or growth in host plants. A recent study revealed that vacuolar amino acid transporters play an important role in spore formation in <i>Schizosaccharomyces pombe</i> and <i>Saccharomyces cerevisiae</i>. To investigate the role of vacuolar amino acid transporters of this phytopathogenic fungus, the <i>FOXG_11334</i> (<i>FoAVT3</i>) gene from <i>F. oxysporum</i> was isolated and its function was characterized. Transcription of <i>FoAVT3</i> was upregulated after rapamycin treatment. A green fluorescent protein fusion of FoAvt3p was localized to vacuolar membranes in both <i>S. cerevisiae</i> and <i>F. oxysporum</i>. Analysis of the amino acid content of the vacuolar fraction and amino acid transport activities using vacuolar membrane vesicles from <i>S. cerevisiae</i> cells heterologously expressing <i>FoAVT3</i> revealed that FoAvt3p functions as a vacuolar amino acid transporter, exporting neutral amino acids. We conclude that the <i>FoAVT3</i> gene encodes a vacuolar neutral amino acid transporter.</p> <p>Localization of vacuolar neutral amino acid transporter, GFP-FoAvt3p, in <i>Fusarium oxysporum</i>. FoAvt3p (green) functions as transporter of neutral amino acids from vacuole (blue) to cytosol.</p

Predicted topology model and intracellular localization of SpAvt3p.

<p>(A)<i>Top</i>, predicted topology model of SpAvt3p. <i>Bottom</i>, sequence alignments of SpAvt3p (Q10074.1) in TM6 (amino acids 451–477) and analogous regions in the homologs according to CLUSTALW: <i>Saccharomyces cerevisiae</i> Avt3p and Avt4p (P36062 and P50944, respectively), <i>Arabidopsis thaliana</i> At5G65990 (ABH04593), and human hPAT1and hPAT2 (AAI36439 and AAI01104, respectively). Identical and similar residues are denoted by <i>black boxes</i> and <i>gray boxes</i>, respectively. The conserved glutamate residue is indicated by an asterisk. (B) The <i>avt3</i>Δ mutant cells expressing GFP-SpAvt3p fusion protein were subjected to fluorescence microscopy. Vacuolar membranes were stained with FM4-64. BF, bright field; bar, 5 μm.</p

Effects of <i>avt3</i>⁺ expression on the ATP-dependent uptake of basic amino acids by vacuolar membrane vesicles.

<p>Vacuolar membrane vesicles were isolated from the wild-type cells carrying an empty plasmid (<i>squares</i>), the <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The amino acid uptake assay was performed with (<i>black symbols</i>) or without (<i>white symbols</i>) 2 mM ATP. The values represent the mean ± SD based on at least three independent experiments.</p

Effects of <i>avt3</i>⁺ expression on the vacuolar amino acid composition of <i>S</i>. <i>cerevisiae</i>.

<p>The vacuolar pools of <i>S</i>. <i>cerevisiae</i> were prepared and analyzed using an amino acid analyzer. The results represent the mean ± SD based on at least three independent experiments: wild-type cells carrying an empty plasmid (<i>white bar</i>), <i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>black bar</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>light gray bar</i>), and pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>dark gray bar</i>).</p

SpAvt3p-dependent extrusion of amino acids by vacuolar membrane vesicles.

<p>(A) Immunoblot analysis of GFP-SpAvt3p and GFP-SpAvt3p^E469A in the vacuolar membrane vesicles isolated from <i>S</i>. <i>cerevisiae avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ mutant cells. Vacuolar membrane vesicles were prepared and analyzed by immunoblotting using anti-GFP serum and anti-Pho8 antibody. Pho8p was detected as the loading control. (B) Alanine and tyrosine export by vacuolar membrane vesicles. [¹⁴C]-labeled amino acids were preloaded into the vacuolar membrane vesicles isolated from <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid (<i>circles</i>), pGPD-GFP-<i>avt3</i>⁺ (<i>triangles</i>), or pGPD-GFP-<i>avt3</i>^<i>E469A</i> (<i>diamonds</i>). The export assay was performed in the presence (<i>black symbols</i>) or absence (<i>white symbols</i>) of 2 mM ATP. Preloaded vesicles were removed immediately before (0 min) or at 1, 2, 4, and 8 min after the addition of ATP, and collected on cellulose acetate membrane filters. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters are shown. The values represent the mean ± SD based on at least three independent experiments. (C) Effects of CCA on ATP-driven alanine and tyrosine export. The experiments were performed as described above. Vacuolar membrane vesicles were incubated with 1 μM CCA for 10 min before loading with [¹⁴C]-labeled amino acids. The amount of preloaded [¹⁴C]-labeled amino acids at 0 min was taken as 100%. The relative amounts trapped on the filters at 8 min after the addition of ATP are shown. The values represent the mean ± SD based on at least three independent experiments: <i>avt1</i>Δ<i>avt3</i>Δ<i>avt4</i>Δ cells carrying an empty plasmid without (<i>white bar</i>) or with (<i>black bar</i>) CCA, and pGPD-GFP-<i>avt3</i>⁺ without (<i>light gray bar</i>) or with CCA (<i>dark gray bar</i>).</p

Vacuolar morphology of <i>S</i>. <i>pombe</i> ARC039 (wild-type), <i>avt3</i>Δ, <i>avt3</i>Δ/pTN54, and <i>avt3</i>Δ/pTN54-<i>avt3</i>⁺.

<p>Cells were grown in MM medium without thiamine for 20 h and the vacuolar membranes were then stained with FM4-64. BF, bright field; bar, 5 μm.</p

Basic native-PAGE patterns for the reconstitution of wild-type/mutant catalytic domains (V₁ domains).

<p>Purified wild-type or mutant A ₃B₃ and DF complexes were mixed in a 1: 5 molar ratio and incubated on ice for 1 h to reconstitute the catalytic domain A ₃B₃DF, as described in Materials and Methods. Lanes 1, 10, 12, and 14: purified wild-type and mutant A ₃B₃ complexes; lane 2: wild-type A ₃B₃DF; lanes 3, 9, 11, 13, 15, 17: reconstituted mutant catalytic domains; and lanes 18 and 19: B and A monomers, respectively. Three micrograms of proteins were loaded in lanes 1, 9, 16, and 17, and 2 µg in lanes 10, 15, 18, and 19.</p

Schematic model of <i>E</i><i>. hirae</i> V-ATPase (adapted from [15] and [16]).

<p>The V₁ domain of V-ATPase is composed of a hexameric arrangement of alternating A and B subunits responsible for ATP binding and hydrolysis; it also contains the DF subunits (shown by a dotted red line), the focus of this study. The V_o domain of V-ATPase comprises an a subunit and an attached membrane c ring. The V₁ and V_o domains are connected by a central stalk, which is composed of D, F, and d subunits, and 2 peripheral stalks assembled from the E and G subunits of V₁. ATP hydrolysis induces the rotation of the central axis (DFd complex) together with the c ring, which causes Na⁺ to be pumped through the channel at the interface between the c ring and the a subunit.</p

ATPase activities and their stability in mutant A₃B₃DF complexes of <i>E</i><i>. hirae</i> V-ATPase.

<p>ATPase activities of the mutants were measured using an ATP regeneration system as described in Materials and Methods. <i>A</i>, ATPase activities of the central-axis D subunit mutants measured using various concentrations of ATP. <i>B</i>, Lineweaver-Burk plots of the ATPase activities from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074291#pone-0074291-g004" target="_blank">Figure 4A</a> used for calculating <i>K</i>_m and <i>V</i>_max values for the D mutants. <i>C</i>-<i>D</i>, Stability of ATPase activities of mutant A ₃B₃DF complexes. ATPase activities were measured in the presence of 1 mM ATP. <i>Filled </i><i>circles</i>, A ₃B₃D(RR^165-6AA) F; <i>open </i><i>diamonds</i>, A ₃B₃D(R¹⁶⁶ A) F; <i>filled </i><i>diamonds</i>, A ₃B₃D(R¹⁶⁵ A) F; <i>filled </i><i>triangles</i>, A ₃B(V³⁸⁸ A) ₃D(RR^165-6AA) F; <i>open </i><i>triangles</i>, A(R⁴⁷⁵ A) ₃B₃D(RR^165-6AA) F; <i>filled </i><i>squares</i>, A ₃B(L³⁸⁹ A) ₃DF; <i>open </i><i>squares</i>, A(R⁴⁷⁵ A) ₃B₃DF; <i>open </i><i>crosses</i>, A ₃B(V³⁸⁸ A) ₃DF; and <i>open </i><i>circles</i>, wild-type A ₃B₃DF.</p