CAREER: Functional Analysis of the Tomato Ca-ATPase Mechanism

职业：番茄 Ca-ATP 酶机制的功能分析

• 批准号: 0347202
• 负责人: Craig Gatto
• 金额: $ 66.95万
• 依托单位: Board of Trustees of Illinois State University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0347202&HistoricalAwards=false
• 关键词: CAREER; Functional; Analysis; Tomato; Ca

The goal of this project is to understand the functional mechanism of plant Calcium-ATPases. Ca-ATPases belong to the ubiquitous protein family of P-type ATPases, which exist in all taxonomic phyla. Although investigations of these transporters began in the late 1950's, studies of plant members have only recently begun to appear. This project aims to identify key structural features involved in nucleotide selectivity and membrane trafficking of the ER-type Ca-ATPase from Lycopersicon esculentum (LCA, i.e. the tomato plant). Interestingly, although animal Ca pumps are obligatory for ATP, it appears that plant Ca pumps have a rather relaxed requirement for nucleoside triphosphates. Since phosphorus has limited availability in soil (thus the need for adding P-containing fertilizers to crops), cytoplasmic ATP levels can fluctuate dramatically. Thus, it is possible that plant pumps have evolved mechanisms to utilize many high-energy phosphate compounds to combat phosphate starvation. This research will compare structure-function differences between tomato LCA and animal sarco/endoplasmic reticulum Ca pump (SERCA) to identify critical contact points in the catalytic center that provide relaxed nucleotide selection without altering cation coupling. An important goal of this project is to determine the structure of the LCA nucleotide-binding domain using X-ray crystallography. This atomic resolution structure would allow direct comparisons to the recent animal SERCA structure. Also, the cellular distribution of plant Ca-pumps remains unresolved. For example, calmodulin-stimulated Ca pumps are not only limited to the plasma membrane and "ER-type" Ca pumps are not restricted to organelles. Thus, an additional goal is to identify the membrane pool for LCA and whether its delivery is regulated via phosphorylation. The goals of this investigation will be achieved by 1) expressing full-length wild-type and mutant LCA constructs in yeast, 2) overexpressing and purifying intracellular domains of LCA from E. coli, and 3) engineering transgenic tomato plants expressing green fluorescent protein-tagged versions of LCA. Broader Impact. This project is designed to actively involve undergraduates from underrepresented groups (principally African-American and Hispanics, but including Native Americans and Pacific Islanders) to make significant contributions to the understanding of plant P-type ATPases. This goal includes enhancing the undergraduate curriculum by developing a two credit hour Introduction to Undergraduate Research course that will actively recruit undergraduate minority students from two core sophomore-level courses (i.e. Genetics and Cell Biology). This laboratory-based course will provide opportunities for critical thinking and scientific discovery. Student participation will be directed by the Principle Investigator in conjunction with doctoral students in the laboratory (also enrolled in the scholar-educator program). In addition to receiving academic credit, this course will be a stepping-stone towards entering a thesis-driven undergraduate research project. To enhance participation, funds are budgeted to support interested students during the summer. Lastly, the undergraduate participants will synergistically join a current outreach effort in the department to bring current scientific investigation to local middle- and high-schools. These activities will enhance Illinois State University retention of underrepresented students and encourage diversity for future applicants.

本项目的目的是了解植物钙ATP酶的功能机制。 Ca-ATP酶属于普遍存在的P-型ATP酶家族，存在于所有的分类门中。 尽管对这些转运蛋白的研究始于20世纪50年代末，但对植物成员的研究只是最近才开始出现。 该项目旨在确定番茄（Lycopersicon esculentum，即番茄植物）ER型Ca-ATPase的核苷酸选择性和膜运输的关键结构特征。 有趣的是，虽然动物钙泵是ATP的必需品，但植物钙泵对核苷三磷酸的需求似乎相当宽松。 由于磷在土壤中的可用性有限（因此需要向作物添加含磷肥料），细胞质ATP水平可能会大幅波动。 因此，植物泵可能已经进化出利用许多高能磷酸盐化合物来对抗磷酸盐饥饿的机制。 本研究将比较番茄LCA和动物肌浆网/内质网钙泵（SERCA）之间的结构-功能差异，以确定催化中心的关键接触点，提供宽松的核苷酸选择而不改变阳离子偶联。 本项目的一个重要目标是使用X射线晶体学确定LCA核苷酸结合结构域的结构。 这种原子分辨率结构将允许与最近的动物SERCA结构进行直接比较。 此外，植物钙泵的细胞分布仍然没有得到解决。 例如，钙调素刺激的Ca泵不仅限于质膜，并且“ER型”Ca泵不限于细胞器。 因此，另一个目标是确定LCA的膜池以及其递送是否通过磷酸化调节。 本研究的目的是通过1）在酵母中表达全长野生型和突变型LCA构建体，2）从E.大肠杆菌，和3）工程化表达LCA的绿色荧光蛋白标记形式的转基因番茄植物。更广泛的影响。 该项目旨在积极参与来自代表性不足的群体（主要是非洲裔美国人和西班牙裔，但包括美洲原住民和太平洋岛民）的本科生，为理解植物P型ATP酶做出重大贡献。 这一目标包括通过开发两个学分的本科研究课程介绍来加强本科课程，该课程将积极招收两个核心预科课程（即遗传学和细胞生物学）的本科少数民族学生。 这个实验室为基础的课程将提供批判性思维和科学发现的机会。 学生的参与将由主要研究者与实验室的博士生一起指导（也参加了研究生教育计划）。 除了获得学分，这门课程将是进入论文驱动的本科研究项目的垫脚石。 为了加强参与，预算资金用于在夏季支持感兴趣的学生。 最后，本科生参与者将协同加入该部门目前的外联工作，将目前的科学调查带到当地的初中和高中。 这些活动将提高伊利诺伊州立大学保留代表性不足的学生，并鼓励未来的申请人的多样性。