透過跨膜螺旋交換探究AtNPF6.3與AtNPF8.1 之轉運蛋白活性

Abstract

氮是植物生長發育的必需營養素。硝酸鹽轉運蛋白1/胜肽轉運蛋白家族（NPF）調控植物對各種含氮化合物的吸收。在此家族成員中，AtNPF6.3負責硝酸鹽轉運，而AtNPF8.1負責胜肽轉運。然而，決定其受質特異性背後的分子機制仍不清楚。 為了了解決定NPF受質特異性的原因，相對於過去的單一胺基酸置換，本研究置換跨膜螺旋(transmembrane helix, TMH)。透過在AtNPF6.3和AtNPF8.1之間交換TMH 1、7和8，產生九個嵌合體。在圓葉菸草葉片和酵母細胞中進行的亞細胞定位分析中，證實所有嵌合蛋白均位在細胞膜上，並透過西方墨點法驗證嵌合蛋白的表現。為了評估硝酸鹽轉運能力，我們利用在本研究中改良的漢遜酵母評估系統，測試嵌合蛋白的硝酸鹽吸收能力。此外，我們也利用釀酒酵母評估系統，測試嵌合蛋白的雙胜肽吸收能力。結果發現所有嵌合蛋白均未表現出吸收硝酸鹽或雙胜肽的能力。這些結果表明，本研究中選擇的TMH交換不會改變受質特異性。

Nitrogen is an essential nutrient for plant growth and development. The Nitrate Transporter 1/Peptide Transporter Family (NPF) mediates the uptake of various nitrogen-containing compounds in plants. Among the NPF family members, AtNPF6.3 is responsible for nitrate transport, while AtNPF8.1 functions in peptide transport. However, the molecular mechanisms underlying their distinct substrate specificities remain unclear. To investigate the determinants of substrate specificity in NPF transporters, this study employed a domain-swapping strategy focused on transmembrane helices (TMH), instead of single-residue substitutions. Nine chimeric constructs were generated by swapping TMH 1, 7, and 8 between AtNPF6.3 and AtNPF8.1. Subcellular localization analyses in Nicotiana benthamiana leaves and yeast cells confirmed that all chimeric proteins were targeted to the plasma membrane. Western blot analysis verified protein expression. To assess substrate transport activity, nitrate uptake was tested in Hansenula polymorpha, using an improved evaluation system developed in this study. Additionally, dipeptide uptake was evaluated in Saccharomyces cerevisiae. None of the chimeric proteins demonstrated functional nitrate or peptide transport activity in either system. These findings suggest that TMH swapping selected in this study cannot alter substrate specificity.