Characterization of an amino acid permease from the endomycorrhizal fungus Glomus mosseae

Abstract

Arbuscular mycorrhizal (AM) fungi are capable of exploiting organic nitrogen sources, but the molecular mechanisms that control such an uptake are still unknown. Polymerase chain reaction-based approaches, bioinformatic tools, and a heterologous expression system have been used to characterize a sequence coding for an amino acid permease (GmosAAP1) from the AM fungus Glomus mosseae. The GmosAAP1 shows primary and secondary structures that are similar to those of other fungal amino acid permeases. Functional complementation and uptake experiments in a yeast mutant that was defective in the multiple amino acid uptake system demonstrated that GmosAAP1 is able to transport proline through a proton-coupled, pH- and energy-dependent process. A competitive test showed that GmosAAP1 binds nonpolar and hydrophobic amino acids, thus indicating a relatively specific substrate spectrum. GmosAAP1 mRNAs were detected in the extraradical fungal structures. Transcript abundance was increased upon exposure to organic nitrogen, in particular when supplied at 2 mm concentrations. These findings suggest that GmosAAP1 plays a role in the first steps of amino acid acquisition, allowing direct amino acid uptake from the soil and extending the molecular tools by which AM fungi exploit soil resources.

Figure 1.

A, GmosAAP1-mediated [¹⁴C]Pro uptake at different substrate concentrations. The experiments were performed at pH 4.5. The values represent means of three independent experiments ± sd. B, pH dependence of the uptake rate of [¹⁴C]Pro in the yeast mutant 22Δ8AA expressing GmosAAP1. Yeast expressing GmosAAP1 in pDR196 were measured at different pH values and an 18.8 μm substrate concentration. The values represent means of three independent experiments ± sd. C, Influence of plasma membrane energization on the uptake rate of [¹⁴C]Pro in the yeast mutant 22Δ8AA expressing GmosAAP1. The yeast cells were preincubated for 5 min in the presence of 100 mm Glc (control), without Glc, or with Glc and 0.1 mm 2,4-dinitrophenol (DNP), 0.1 mm diethylstilbestrol (DES), 0.1 mm carbonyl cyanide m-chlorophenylhydrazone (CCCP), or 0.1 mm vanadate. The values represent means of three independent experiments ± sd.

Figure 2.

Substrate specificity of GmosAAP1. Inhibition of 18.8 μm [¹⁴C]Pro uptake by a 5-fold molar excess of competing amino acids. The data are expressed as percentages of the uptake rate in the presence of 18.8 μm Pro. The values represent means of three independent experiments ± sd.

Figure 3.

Gel electrophoresis of RT-PCR products obtained with oligonucleotides specific for the G. mosseae 28S rDNA (A) or GmosAAP1 (B) on the following samples: lane 1, germinating spores; lane 2, extraradical mycelium; lane 3, intraradical mycelium; lane 4, no template.

Figure 4.

Gel electrophoresis of RT-PCR products obtained with oligonucleotides specific for the G. mosseae 28S rDNA (A) or GmosAAP1 (B) on external mycelium treated as follows: lane 1, no N (0 m); lane 2, 2 μm of the amino acid pool (Leu, Ala, Asn, Lys, and Tyr); lane 3, 2 mm of the amino acid pool; lane 4, 2 μm KNO₃; lane 5, 2 mm KNO₃; lane 6, 2 μm (NH₄)₂SO₄; lane 7, 2 mm (NH₄)₂SO₄.

Figure 5.

Real-time RT-PCR analysis of the GmosAAP1 mRNA in extraradical mycelium treated with 0 m N or 2 μm or 2 mm of the amino acid pool. Relative expression levels were obtained with the comparative threshold cycle method (see “Materials and Methods” for details) and were normalized with respect to the GmosAAP1 levels in the 0 m treatment.