Suppressor of K+ transport growth defect 1 (SKD1) interactswith RING-type ubiquitin ligase and sucrose non-fermenting1-related protein kinase (SnRK1) in the halophyte ice plant

SKD1 (suppressor of K+ transport growth defect 1) is an AAA-type ATPase that functions as a molecular motor. It was previously shown that SKD1 accumulates in epidermal bladder cells of the halophyte Mesembryanthemum crystallinum. SKD1 knock-down Arabidopsis mutants showed an imbalanced Na+/K+ ratio under salt stress. Two enzymes involved in protein post-translational modifications that physically interacted with McSKD1 were identified. McCPN1 (copine 1), a RING-type ubiquitin ligase, has an N-terminal myristoylation site that links to the plasma membrane, a central copine domain that interacts with McSKD1, and a C-terminal RING domain that catalyses protein ubiquitination. In vitro ubiquitination assay demonstrated that McCPN1 was capable of mediating ubiquitination of McSKD1. McSnRK1 (sucrose non-fermenting 1-related protein kinase) is a Ser/Thr protein kinase that contains an N-terminal STKc catalytic domain to phosphorylate McSKD1, and C-terminal UBA and KA1 domains to interact with McSKD1. The transcript and protein levels of McSnRK1 increased as NaCl concentrations increased. The formation of an SKD1–SnRK1–CPN1 ternary complex was demonstrated by yeast three-hybrid and bimolecular fluorescence complementation. It was found that McSKD1 preferentially interacts with McSnRK1 in the cytosol, and salt induced the re-distribution of McSKD1 and McSnRK1 towards the plasma membrane via the microtubule cytoskeleton and subsequently interacted with RING-type E3 McCPN1. The potential effects of ubiquitination and phosphorylation on McSKD1, such as changes in the ATPase activity and cellular localization, and how they relate to the functions of SKD1 in the maintenance of Na+/K+ homeostasis under salt stress, are discussed

Characterization of bladder cell-specific mcSKD1 and its interacting proteins in halophyte Mesembryanthemum crystallinum L.

冰花(Mesembryanthemum crystallinum L.)是一種具有C3-CAM光合作用轉換形式植物可生存於高鹽環境的耐鹽模式植物，表皮特化的腎型細胞在高鹽環境下液胞會迅速膨大，具有儲存水分及區隔過量鈉離子的功能。利用酸性下會呈紅色的染劑neutral red浸染不同時期的冰花表皮細胞，觀察到冰花腎型細胞液胞pH值除了會隨著發育逐漸由酸性轉變為中性外，幼年期的腎型細胞在照光時液胞pH值亦會快速升高，顯示腎型細胞的代謝旺盛並非僅供儲存之用，還具有調節離子平衡之功能。冰花的mcSKD1 (suppressor of K+ transport growth defect)是一個會在冰花的腎型細胞表現的鹽誘導基因，mcSDK1蛋白與酵母菌的液胞運送相關蛋白VPS4 (vacuolar protein sorting 4)和阿拉伯芥AAA-type ATPase (ATPase associated with a variety of cellular activities)具有高度的相似性，並觀察到mcSKD1主要分布在冰花細胞質之ER/Golgi network。為了更進一步瞭解mcSKD1蛋白之功能，以酵母菌雙雜交系統分別由阿拉伯芥基因庫中篩選出與SKD1具有蛋白質交互作用的過氧化逆境相關的catalase 3、細胞壁結構的AGP21 (arabinogalactan protein 21)等蛋白。以及由冰花根部加鹽處理三天所製成的基因庫篩選出與SKD1具有蛋白質交互作用蛋白mcSNF1 (sucrose non-fermenting 1)及mcCPN1 (copine 1)等蛋白。SNF1已知為一參與醣類代謝及逆境相關訊息傳遞作用之蛋白激酶，mcSNF1還具有UBA (ubiquitin associated) domain，mcCPN1蛋白具有與細胞膜蛋白質運送相關之VWA domain及具有E3 ubiquitin ligase活性之RING domain，mcSNF1與mcCPN1也會受加鹽誘導提高蛋白累積量。免疫共沈澱證明了mcSKD1與mcSNF1與mcCPN1具有蛋白質交互作用，以pull-down的蛋白產物比例計算發現，加鹽處理會使mcSKD1與mcSNF1結合的比例提高三倍。利用蔗糖梯度分離microsome蛋白以及細胞免疫染色法，發現加鹽處理會快速改變mcSKD1、mcSNF1和mcCPN1在細胞中分佈的位置，且colocolization的比例提高。當以破壞細胞骨架的藥劑處理細胞時，mcSKD1及mcSNF1在細胞中的分布改變，累積在細胞邊緣，推測細胞骨架參與mcSKD1在細胞中的運輸。鹽處理會造成阿拉伯芥snf1或copine1突變株中atSKD1不正常的聚集，並對鹽更加敏感。實驗結果推測冰花mcSKD1蛋白透過與逆境訊息傳遞mcSNF1、細胞膜相關蛋白mcCPN1的交互作用，參與鹽逆境時ubiquitin相關的蛋白質運送或是逆境訊息傳遞，幫助耐鹽植物冰花在高鹽環境下維持正常生理代謝。The halophyte Mesembryanthemum crystallinum L. (ice plant) is an inducible CAM and a model plant for studying salt-tolerant mechanisms in higher plants. It contains specialized epidermal bladder cells (EBCs) which rapidly expend under salt stress. The major functions of EBCs are maintaining ion homeostasis and water storage. During plant development, increase in vacuolar pH was observed in EBCs. In addition, light-induced rapid change in vacuolar pH was found in juvenile stage of ice plant indicating EBCs are metabolic active cells. A salt-induced gene mcSKD1 (suppressor of K+ transport growth defect) is highly expressed in EBCs. It has high homology to yeast VPS4 (vacuolar protein sorting 4) and Arabidopsis AAA-type ATPase (ATPase associated with a variety of cellular activities). Immunofluorescence labeling showed mcSKD1 protein was located in cytoplasm around ER/Golgi network. Yeast two-hybrid screen was performed to identify mcSKD1-interacting proteins. Using a library constructed from Arabidopsis, catalase 3 and AGP21 (arabinogalactan protein 21) were identified. Using a library constructed from salt-treated ice plant roots, mcSNF1 (sucrose non-fermenting 1) and mcCPN1 (copine 1) were further characterized. Yeast SNF1 is a ser/thr kinase that plays an important role in carbon metabolism and stress signaling. The SNF1 protein identified in ice plant contained an extra UBA (ubiquitin associated) domain. Sequence analysis of mcCPN1 showed it contains a VWA domain for membrane trafficking and a RING domain for protein ubiquitination. The accumulation of mcSNF1 and mcCPN1 was both induced by salt stress. Co-immunoprecipitation experiment showed mcSKD1 interacted with both mcSNF1 and mcCPN1 in vitro. In vivo pull-down assay showed a 3-fold increase in the association between mcSKD1 and mcSNF1 under salt stress. Microsomal fractionation and immunolabeling experiments showed salt induced rapid changes in cellular localization of mcSKD1, mcSNF1, and mcCPN1. When the cytoskeleton was disrupted, the distribution of mcSKD1 and mcSNF1 was altered, as seen by abnormal aggregation around plasma membrane. The result suggested that mcSKD1 is trafficking along the cytoskeleton. Arabidopsis snf1 and copine 1 mutants showed more salt sensitive than the wild type and aggregation of atSKD1 inside mutant cells. The results suggested that mcSNF1 and mcCPN1 function together with mcSKD1 in ubiquitin-related protein trafficking and stress signal transduction in order to maintain normal growth under high salinity.Catalog: Abstract in Chinese II Abstract IV Catalog VI Catalog of tables VIII Catalog of figures IX Preface 1 Chapter 1 6 Abstract 7 Introduction 8 Material and methods 10 Results 12 Discussion 14 References 17 Chapter 2 24 Introduction 25 Material and methods 30 Results 34 Discussion 39 References 45 Chapter 3 73 Introduction 74 Material and methods 78 Results 84 Discussion 91 References 95 Catalog of tables: Chapter 2 Table 1. The category “tissue” was used in similarity search. Following genes are sorted with AtSKD1…………………………………………………. 54 Table 2. The category “nutrient treatment” was used in similarity search. Following genes are sorted with AtSKD1………………………………… 55 Table 3. The category “abiotic stress” was used in similarity search. Following genes are sorted with AtSKD1…………………………………………….. 56 Table 4. Putative mcSKD1-interacting proteins identified from Arabidopsis library…………………………………………………………………….. 57 Table 5. Putative mcSKD1-interacting proteins identified from salt-stressed ice plant library……………………………………………………………….. 58 Chapter 3 Table 1. Search mcSKD1 protein modification pattern on PROSITE database……………………………………………………………….. 102 Table 2. Search mcSNF1 protein modification pattern on PROSITE database………………………………………………………………... 103 Table 1. Search mcCPN1 protein modification pattern on PROSITE database………………………………………………………………... 104 Catalog of figures: Chapter 1 Fig. 1. Morphology and vacuole acidity of ice plant EBC during development... 21 Fig 2. The light-induced decrease in acidity of EBCs…………………………... 22 Fig 3. Change in composition of raphide crystals……………………………….. 23 Chapter 2 Fig 1. Quantitative analysis of atSKD1 expression under various stresses……... 60 Fig 2. Total cellular protein extracted from 10-day-old cultures ice plant cells… 61 Fig 3. Distribution of mcSKD1 in microsomal fractions………………………... 62 Fig 4. Double-labeling immunofluorescence……………………………………. 63 Fig 5. Immunstaining of ice plant leaf EBCs……………………………………. 64 Fig 6. The accumulation of atSKD1 in 8-week-old Arabidopsis plants…………. 65 Fig 7. The localization of atSKD1 in cultured Arabidopsis cells………………... 66 Fig 8. Isolation of Poly A+ mRNA for yeast two hybrid screening library……… 67 Fig 9. In vivo interaction between mcSKD1 and Catalase 3 or AGP21………… 68 Fig 10. In vivo interaction between mcSKD1 and Copine 1 or SNF-1…………. 69 Fig 11. Arabidopsis QTL markers showing positions of SKD1-interaction proteins…………………………………………………………………… 70 Fig 12. Characterization of the mcSNF1 from ice plant………………………… 71 Fig 13. Characterization of the mcCPN1 from ice plant………………………… 72 Chapter 3 Fig 1. Time course expression of mcSNF1in plant callus under salt stress……... 105 Fig 2. Time course expression of mcCPN1 under salt stress…………………… 106 Fig 3. In vitro coimmunoprecipitation of mcSKD1 with mcCPN1 or mcSNF1… 107 Fig 4. The salt stress changed protein-protein interaction ratio of mcSKD1, mcSNF1 and mcCPN1.…………………………………………………… 108 Fig 5. Double-labeling immunofluorescence of mcSKD1 and mcCPN1 in ice plant cells.………………………………………………………………… 109 Fig 6. Double-labeling immunofluorescence of mcSKD1 and mcSNF1 in ice plant cells………………………………………………………………… 110 Fig 7. Time-course accumulation of mcSKD1, mcSNF1 and mcCPN1 within 24 hours of salt stress……………………………………………………….. 111 Fig 8. mcSNF1 is a extracellular protein.………………………………………. 112 Fig 9. Analysis of protein modification…………………………………………. 113 Fig 10. Distribution of mcSKD1, mcSNF1 and mcCPN1 in microsomal fractions of ice plant leaves.……………………………………………… 114 Fig 11. Distribution of mcSKD1, mcSNF1, and mcCPN1 in microsomal fractions.………………………………………………………… 115 Fig 12. Cell viability test………………………………………………………… 116 Fig 13. Initial changes in cellular distribution of mcSKD1, mcSNF1, and mcCPN1 by 200 mM NaCl.………………………………………………. 117 Fig 14. The effects of cytoskeleton-disrupting drugs on mcSDK1 distribution and endocytosis pathway in ice plant cells.……………………………. 118 Fig 15. The effects of cytoskeleton-disrupting drugs on mcSKD1, mcSNF1 and mcCPN1 localization……………………………………………………………. 119 Fig 16. Accumulation of atSKD1 protein in wild type and akin10, copine1 mutants.………………………………………………………………… 120 Figure 17. Dose-dependent accumulation of atSKD1 protein in wild type Arabidopsis and copine1, snf1 mutants.………………………………….. 121 Figure 18. Double-labeling immunofluorescence on wild type Arabidopsis, snf1, and copine1 mutants……………………………………………. 12