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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 鄭貽生 | zh_TW |
| dc.contributor.advisor | Yi-Sheng Cheng | en |
| dc.contributor.author | 曾祥弘 | zh_TW |
| dc.contributor.author | Hsiang-Hung Tseng | en |
| dc.date.accessioned | 2025-08-21T16:38:05Z | - |
| dc.date.available | 2025-08-22 | - |
| dc.date.copyright | 2025-08-21 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-07-30 | - |
| dc.identifier.citation | Akter M, Qin C, Malik A.I (2015). OsNPF2.4 mediates low-affinity nitrate uptake. J. Exp. Bot 66, 317–327.
Biegel A, Gebauer S, Hartrodt B, Brandsch M, Neubert K (2006). The renal type H⁺/peptide symporter PEPT2: structure-affinity relationships. J. Amino Acids 31(2), 137–156. Cregg J.M, Vedvick T.S, Raschke W.C (1993). Recent advances in the expression of foreign genes in Pichia pastoris. Biotechnol. J 11(8), 905–910. Dechorgnat J, Patrit O, Krapp A, Daniel-Vedele F (2012). Characterization of the nitrate transporter NRT2.6 in Arabidopsis thaliana. Plant Mol Biol 79(3), 239–251. Deng J, Wu Y, Zheng Z, Chen N, Luo X, Tang H.T, Keasling J.D (2021). A synthetic promoter system for well-controlled protein expression with different carbon sources in Saccharomyces cerevisiae. Microb Cell Fact 20, 202 Dietrich D, Hammes U, Thor K, Suter-Grotemeyer M, Flückiger R, Slusarenko A. J, Ward J.M, Rentsch D (2004). AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis. Plant J 40(4), 488–499. Doki S, Kato H.E, Solcan N, Iwaki M, Koyama M, Hattori M, Iwase N, Tsukazaki T, Sugita Y, Kandori H, Newstead S, Ishitani R, Nureki O (2013) Structural basis for dynamic mechanism of proton-coupled symport by the peptide transporter POT, Proc. Natl. Acad. Sci. U.S.A. 110 (28), 11343-11348 Feng H, Xia X, Fan X, Xu G, Miller A.J (2013). Optimizing plant transporter expression in Xenopus oocytes. Plant Methods 9(1), 48. Filleur S, Dorbe M.F, Cerezo M, Orsel M, Granier F, Gojon A, Daniel-Vedele F (2001). An Arabidopsis T-DNA mutant affected in Nrt2 genes is impaired in nitrate uptake. FEBS Lett 489(2–3), 220–224. Gellissen G, Janowicz Z.A, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg C.P, Strasser A.W (1991). Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Biotechnol. J 9(3), 291–295. Gellissen G, Janowicz Z.A, Weydemann U, Melber K, Strasser A.W, Hollenberg C.P (1992). High-level expression of foreign genes in Hansenula polymorpha. Biotechnol Adv 10(2), 179–189. Guan Y, Liu D.F, Qiu J, Liu Z.J, He Y.N, Fang Z.J, Huang X.H, Gong J.M (2022). The nitrate transporter OsNPF7.9 mediates nitrate allocation and the divergent nitrate use efficiency between indica and japonica rice. Plant Physiol 189(1), 215-229. Guettou F, Quistgaard E.M, Trésaugues L, Moberg P, Jegerschöld C, Zhu L, Jong A.J, Nordlund P, Löw C (2013). Structural insights into substrate recognition in proton-dependent oligopeptide transporters. EMBO Rep 14(9), 804–810. Hammes UZ, Meier S, Dietrich D, Ward JM, Rentsch D (2010) Functional properties of the Arabidopsis peptide transporters AtPTR1 and AtPTR5. J Biol Chem 285(51), 39710-7. Ho C.H, Lin S.H, Hu H.C, Tsay Y.F (2009). CHL1 functions as a nitrate sensor in plants. Cell 138(6), 1184–1194. Huang N.C, Liu K.H, Lo H.J, Tsay Y.F (1996). Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell 8(12), 2183–2191. Huyer G, Piluek W.F, Fansler Z, Kreft S.G, Hochstrasser M, Brodsky J.L, Michaelis S (2004). Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. JBC 279(37), 38369–38378. Kanstrup C, Nour-Eldin H.H (2022). The emerging role of the nitrate and peptide transporter family: NPF in plant specialized metabolism. Curr. Opin. Plant Biol 68, 102243. Komarova N.Y, Thor K, Gubler A, Meier S, Dietrich D, Weichert A, Grotemeyer M.S, Tegeder M, Rentsch D (2008). AtPTR1 and AtPTR5 transport dipeptides in planta. Plant Physiol 148(2), 856–869. Lauwers E, Jacob C, André B (2010). K63-linked ubiquitin chains as a specific signal for protein sorting into the multivesicular body pathway. J Cell Biol 185(3), 493–502. Léran S, Varala K, Boyer J.C, Chiurazzi M, Crawford N, Daniel-Vedele F, David L, Dickstein R, Fernandez E, Forde B, Gassmann W, Geiger D, Gojon A, Gong J.M, Halkier B.A, Harris J.M, Hedrich R, Limami A.M, Rentsch D, Seo M, Tsay Y.F, Zhang M.Y, Coruzzi G, Lacombe B (2014). A unified nomenclature of NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER family members in plants. Trends Plan Sci 19(1), 5–9. Liang R, Fei Y.J, Prasad P.D, Ramamoorthy S, Han H, Yang-Feng T.L, Hediger M.A, Ganapathy V, Leibach F.H (1995). Human intestinal H+/peptide cotransporter. Cloning, functional expression, and chromosomal localization. J Biol Chem 270(12), 6456–6463. Lillemeier B.F, Pfeiffer J.R, Surviladze Z, Wilson B.S, Davis M.M (2006). Plasma membrane-associated proteins are clustered into islands attached to the cytoskeleton. Proc Natl Acad Sci USA 103(50), 18992–18997. Limon A, Mattei C The Xenopus Oocyte: A Tool for Membrane Biology. (2023). Memb 13, 831. Liu K.H, Huang C.Y, Tsay Y.F (1999). CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11(5), 865–874. Liu K.H, Tsay Y.F (2003). Switching between the two action modes of the dual-affinity nitrate transporter CHL1 by phosphorylation. EMBO J 22(5), 1005–1013. Mayer M, Schaaf G, Mouro I, Lopez C, Colin Y, Neumann P, Cartron J.P, Ludewig U (2006). Different transport mechanisms in plant and human AMT/Rh-type ammonium transporters. JBC 281(8), 4940–4947. Miller A.J, Zhou J.J (2000). Xenopus oocytes as an expression system for plant transporters. BBA-biomembranes 1465, 343-358 Mulligan C, Mindell J.A (2013). Mechanism of transport modulation by an extracellular loop in an archaeal excitatory amino acid transporter (EAAT) homolog. J Biol Chem 288(49), 35266-76. Newstead S (2015). Molecular insights into proton coupled peptide transport in the PTR family of oligopeptide transporters. BBA 1850(3), 488–499. Newstead S, Drew D, Cameron A.D, Postis V.L.G, Xia X, Fowler P.W, Ingram J.C, Carpenter E.P, Sansom M.S.P, McPherson M.J, Baldwin S.P, Iwata S (2011). Crystal structure of a prokaryotic homologue of the mammalian oligopeptide-proton symporters, PepT1 and PepT2. EMBO J 30(2), 417–426. Nour-Eldin H.H, Nørholm M.H.H, Halkier B.A (2006). Screening for plant transporter function by expressing a normalized Arabidopsis cDNA library in Xenopus oocytes. Plant Methods 2, 17. Parker J.L, Newstead S (2014). Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature 507(7490), 68–72. Partow S, Siewers V, Bjørn S, Nielsen J, Maury J (2010). A new platform host for strong expression under GAL promoters without inducer in Saccharomyces cerevisiae. Microb. Cell Fact 9, 57. Pike S.M, Matthes M, McSteen P, Gassmann W (2019). Using Xenopus laevis oocytes to functionally characterize plant transporters. Curr Protoc Plant Biol 4(2), e20087 Podolsky I.A, Seppälä S, Xu H, Jin Y.S, O'Malley M.A (2021). A SWEET surprise: Anaerobic fungal sugar transporters and chimeras enhance sugar uptake in yeast. Metab. Eng. 66, 137–147. Schmidt F.C, Fitz K, Feilen L.P, Okochi M, Steiner H, Langosch D (2023). Different transmembrane domains determine the specificity and efficiency of the cleavage activity of the γ-secretase subunit presenilin. J. Biol. Chem 299(5), 104626. Solcan N, Kwok J, Fowler P.W, Cameron A.D, Drew D, Iwata S, Newstead S (2012). Alternating access mechanism in the POT family of oligopeptide transporters. EMBO J 31(2), 341–355. Stacey G, Koh S, Granger C, Becker J.M (2002). Peptide transport in plants. Trends Plant Sci 7(6), 257–263. Stauffer M, Jeckelmann J.M, Ilgü H, Ucurum Z, Boggavarapu R, Fotiadis D (2022). Peptide transporter structure reveals binding and action mechanism of a potent PEPT1 and PEPT2 inhibitor. Commun Chem 5(1), 23. Sun J, Bankston J.R, Payandeh J, Hinds T.R, Zagotta W.N, Zheng N (2014). Crystal structure of the plant dual-affinity nitrate transporter NRT1.1. Nature 507(7490), 73–77. Sun J, Zheng N (2015). Molecular mechanism underlying substrate recognition and transport by the plant nitrate transporter NRT1.1. Cell Res 25(5), 504–507. Terada T, Irie M, Okuda M, Katsura T, Inui K (2000). Expression profiles of peptide transporters in epithelial tissues. Biochem. Biophys. Res. Commun 252(3), 698–702. Tsay Y.F, Chiu C.C, Tsai C.B, Ho C.H, Hsu P.K (2007). Nitrate transporters and peptide transporters. FEBS Lett. 581(12), 2290–2300. Tsay Y.F, Schroeder J.I, Feldmann K.A, Crawford N.M (1993). The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible nitrate transporter. Cell 72(5), 705–713. Zhou C, Slaughter B.D, Unruh J.R, Guo F, Yu Z, Mickey K, Narkar A, Ross R.T, McClain M, Li R (2014). Organelle-based aggregation and retention of damaged proteins in asymmetrically dividing cells. Cell 159(3), 530–542. 康峰誌 (2021). 闡述 AtNPF8.1/AtPTR1 受質專一性的分子特徵. 臺灣大學植物科學研究所學位論文, 1-97 張皓羽 (2021). 由分子特徵探討硝酸鹽轉運蛋白 NPF6.3/NRT1.1/CHL1 的受質專一性. 臺灣大學植物科學研究所學位論文, 1-91 | - |
| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/99163 | - |
| dc.description.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。 | zh_TW |
| dc.description.abstract | 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. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-08-21T16:38:05Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-08-21T16:38:05Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | 口試委員會審定書 I
誌謝 II 摘要 III Abstract IV Contents VI List of figures XI List of supplemental figures XII List of tables XIII 1. Introduction 1 1.1 Previous studies of Nitrate 1/Peptide Transporter Family (NPF) 1 1.1.1 AtNPF6.3: structural basis and phosphorylation-dependent regulation of dual-affinity nitrate transport 1 1.1.2 AtNPF8.1: functional characterization and structural insights into proton-coupled peptide transport 2 1.1.3 Identification of key residues and domain-swapping strategy to investigate substrate specificity in AtNPF6.3 and AtNPF8.1 4 1.2 Advantages and limitations of the Xenopus laevis oocyte system for functional analysis of plant NPF transporters 5 1.3 Yeast complementation as a versatile tool for functional and substrate specificity analysis of plant transporters 7 1.4 Hansenula polymorpha as a platform for membrane protein expression and the role of plasmid copy number in functional complementation 8 1.5 Exploring the role of TMH in substrate specificity through domain swapping 9 2. Research aims 11 3. Materials and Methods 12 3.1. Plasmid extraction 12 3.2. Construction of chimeric constructs assembled from more than three fragments 13 3.3. Construction of chimeric constructs assembled from three or fewer fragments 14 3.4. E. coli. transformation 15 3.5. Gel extraction and PCR product purification 16 3.6. Subcloning 16 3.7. Yeast transformation 17 3.8. Yeast nitrate uptake complementation and subcellular localization 19 3.9. Yeast dipeptide uptake complementation and subcellular localization 19 3.10. Yeast protein extraction and Western blot 20 3.11. Agrobacterium transformation 22 3.12. Tobacco growth conditions and agrobacterium infiltration 23 3.13. Colony PCR 24 4. Results 26 4.1 Relative integrated plasmid copies number influences nitrate uptake ability in H. polymorpha 26 4.2 All nine chimeric proteins were localized to the plasma membrane in N. benthamiana leaf cells 26 4.3 All nine chimeric proteins were localized to the plasma membrane in H. polymorpha 27 4.4 All nine chimeric proteins were localized to the plasma membrane in S. cerevisiae 28 4.5 Western blot analysis of chimeric protein expression in H. polymorpha 29 4.6 Western blot analysis of chimeric protein expression in S. cerevisiae 29 4.7 None of the nine chimeric proteins exhibited detectable nitrate uptake activity in H. polymorpha 30 4.8 None of the nine chimeric proteins exhibited detectable dipeptide uptake activity in S. cerevisiae 31 5. Discussion 33 5.1 Assessing colony PCR relative band intensity as an indicator of integrated plasmid copy number 33 5.2 Subcellular localization and potential degradation of chimeric proteins in yeast 33 5.3 Optimization of Western Blot conditions and interpretation of GFP-tagged chimera expression 34 5.4 Evaluating growth curves and determining the nitrate concentration threshold for uptake evaluation in H. polymorpha 35 5.5 Structural modeling reveals potential causes of inactivity in chimeric proteins 36 5.6 Sequence insertions and deletions in TMH2 likely disrupt chimera function 37 5.7 Interpretation of GFP aggregation and peripheral localization patterns in yeast 38 5.8 Conclusion 39 6. Figures 41 7. Supplemental figures 68 8. Tables 83 9. References 84 10. Appendix 88 10.1. Appendix A: Buffer 88 10.2. Appendix B: Primer list 92 10.2. Appendix C: Substrate binding site 94 10.2. Appendix D: Phylogenetic tree 95 List of figures Figure 1. The sequence alignment and schematic diagram of the wild-type AtNPF6.3 (CHL1) and AtNPF8.1 (PTR1), and nine chimeric proteins 42 Figure 2. Correlation of relative integrated plasmid numbers and nitrate uptake ability 45 Figure 3. Localization of chimeric proteins in N. benthamiana 49 Figure 4. Localization of chimeric proteins in yeast H. polymorpha 51 Figure 5. Localization of chimeric proteins in yeast S. cerevisiae. 53 Figure 6. Western analysis of the yeast H. polymorpha. 55 Figure 7. Western analysis of the yeast S. cerevisiae 57 Figure 8. Evaluation of nitrate uptake activity in H. polymorpha expressing chimera-GFP constructs 58 Figure 9. Evaluation of nitrate uptake activity in H. polymorpha expressing chimera without GFP constructs 60 Figure 10. Evaluation of peptide uptake activity in S. cerevisiae expressing chimera-GFP constructs 63 Figure 11. Evaluation of peptide uptake activity in S. cerevisiae expressing chimera without GFP constructs 66 List of supplemental figures Supplemental Figure 1. Assessing colony PCR relative band intensity as an indicator of integrated plasmid copy number 68 Supplemental Figure 2. Attempts for optimization to Western analysis in H. polymorpha and S. cerevisiae 70 Supplemental Figure 3. Growth curve confirms nitrate uptake ability in H. polymorpha 71 Supplemental Figure 4. Determining the nitrate concentration threshold for uptake evaluation in H. polymorpha 73 Supplemental Figure 5. Structural prediction reveals aberrant loops in TMH 2 of specific chimeric proteins 75 Supplemental Figure 6. Repeat of the correlation between integrated plasmid copy number and nitrate uptake ability (Figure 2) 78 Supplemental Figure 7. The sequence alignment of the peptide transporters, nine chimeric proteins, and nitrate transporters 81 List of tables Table 1. RMSD values of chimeric proteins versus AtNPF6.3 83 | - |
| dc.language.iso | en | - |
| dc.subject | AtNPF6.3 | zh_TW |
| dc.subject | 跨膜螺旋交換 | zh_TW |
| dc.subject | AtNPF8.1 | zh_TW |
| dc.subject | 漢遜酵母 | zh_TW |
| dc.subject | Hansenula polymorpha | en |
| dc.subject | Transmembrane helix swapping | en |
| dc.subject | AtNPF8.1 | en |
| dc.subject | AtNPF6.3 | en |
| dc.title | 透過跨膜螺旋交換探究AtNPF6.3與AtNPF8.1 之轉運蛋白活性 | zh_TW |
| dc.title | Functional study on transporter activities of AtNPF6.3 and AtNPF8.1 via transmembrane helix swapping | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-2 | - |
| dc.description.degree | 碩士 | - |
| dc.contributor.coadvisor | 王雅筠 | zh_TW |
| dc.contributor.coadvisor | Ya-Yun Wang | en |
| dc.contributor.oralexamcommittee | 李勇毅;李金美 | zh_TW |
| dc.contributor.oralexamcommittee | Yung-I Lee;Chin-Mei Lee | en |
| dc.subject.keyword | 漢遜酵母,AtNPF6.3,AtNPF8.1,跨膜螺旋交換, | zh_TW |
| dc.subject.keyword | Hansenula polymorpha,AtNPF6.3,AtNPF8.1,Transmembrane helix swapping, | en |
| dc.relation.page | 95 | - |
| dc.identifier.doi | 10.6342/NTU202502822 | - |
| dc.rights.note | 同意授權(限校園內公開) | - |
| dc.date.accepted | 2025-08-01 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 生命科學系 | - |
| dc.date.embargo-lift | 2025-08-22 | - |
| 顯示於系所單位： | 生命科學系 | |
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