Low and high affinity amino acid H⁺‐cotransporters for cellular import of neutral and charged amino acids

Abstract

SummaryAmides and acidic amino acids represent the major long distance transport forms of organic nitrogen. Six amino acid permeases (AAPs) from Arabidopsis mediating transport of a wide spectrum of amino acids were isolated. AAPs are distantly related to plasma membrane amino acid transport systems N and A and to vesicular transporters such as VGAT from mammals. A detailed comparison of the properties by electrophysiology after heterologous expression in Xenopus oocytes shows that, although capable of recognizing and transporting a wide spectrum of amino acids, individual AAPs differ with respect to speci®city. Apparent substrate af®nities are in¯uenced by structure and net charge and vary by three orders of magnitude. AAPs mediate cotransport of neutral amino acids with one proton. Uncharged forms of acidic and basic amino acids are cotransported with one proton. Since all AAPs are differentially expressed, different tissues may be supplied with a different spectrum of amino acids. AAP3 and AAP5 are the only transporters mediating ef®cient transport of the basic amino acids. In vivo competition shows that the capability to transport basic amino acids in planta might be overruled by excess amides and acidic amino acids in the apoplasm. With the exception of AAP6, AAPs do not recognize aspartate; only AAP6 has an af®nity for aspartate in the physiologically relevant range. This property is due to an overall higher af®nity of AAP6 for neutral and acidic amino acids. Thus AAP6 may serve a different role either in cooperating with the lower af®nity systems to acquire amino acids in the low concentration range, as a system responsible for aspartate transport or as an uptake system from the xylem. In agreement, a yeast mutant de®cient in acidic amino acid uptake at low aspartate concentrations was complemented only by AAP6. Taken together, the AAPs transport neutral, acidic and cationic amino acids, including the major transport forms, i.e. glutamine, asparagine and glutamate. Increasing proton concentrations strongly activate transport of amino acids. Thus the actual apoplasmic concentration of amino acids and the pH will determine what is transported in vivo, i.e. major amino acids such as glutamine, asparagine, and glutamate will be mobilized preferentially.

High Affinity Amino Acid Transporters Specifically Expressed in Xylem Parenchyma and Developing Seeds of Arabidopsis

“…In principal most amino acids were good competitors with the exception of the basic amino acids arginine, histidine, and lysine. The selectivity of the competition experiments is in agreement with previous studies showing that the basic amino acids are recognized only by AAP3 and AAP5 in in vivo competition experiments (14). Thus AAP8 can be classified as a general amino acid permease and, when expressed in yeast, mediates a higher transport capacity for aspartate as compared with the other AAPs.…”

“…Aspartate is transported by most AAPs with an extremely low affinity and efficiency. Only AAP6 has a K m value for aspartate that is in a physiological range (14). The described properties of AAPs thus match the activities expected for transport proteins involved in moving a wide spectrum of amino acids into the phloem sap and are consistent with the properties described for uptake kinetics measured in plasma membrane vesicles of leaves (17).…”

“…AAP1 and AAP6 in pFL61 were used for comparison since they are most closely related to AAP8 on protein level. AAP5 was included due to the higher affinity toward basic amino acids (14). On media containing 1 and 3 mM arginine, citrulline, glutamate, or proline as sole nitrogen sources, respectively, cells expressing either of the AAPs were able to grow, except AAP7 (Fig.…”

“…Among the different transporter genes identified in Arabidopsis so far, AAP1 and AAP2 were most highly expressed in immature siliques of Arabidopsis, indicating a role in supplying developing seeds with organic nitrogen. Biochemical properties of these transporters characterize them as low affinity transport systems (14). In analogy to sucrose transport-* This work was supported by the DFG Projects Aminosä uretransport and Schwerpunkt Membrantransport SPP1108 (FR989/10 -1).…”

“…Six of these eight genes have been characterized as functional transporters so far. AAP1-5 were shown to mediate transport of a broad spectrum of amino acids with low selectivity by a protoncoupled mechanism (14,22,28,29). For glutamine, glutamate, and asparagine as major transport forms of organic nitrogen, these transporters have K m values that are in a physiological range with respect to the concentrations of these amino acids found in the phloem and xylem.…”