CAREER: Functional Analysis of the Tomato Ca-ATPase Mechanism

本项目旨在了解植物钙- atp酶的功能机制。ca - atp酶属于普遍存在的p型atp酶蛋白家族，存在于所有分类门中。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直到最近才开始出现。本项目旨在确定番茄（LCA，即番茄植物）er型ca - atp酶的核苷酸选择性和膜运输的关键结构特征。有趣的是，尽管动物钙泵对ATP是必需的，但植物钙泵对三磷酸核苷的要求似乎相当宽松。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水平可能会剧烈波动。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sarco/内质网Ca泵（SERCA）的结构-功能差异，以确定催化中心的关键接触点，这些接触点在不改变阳离子偶联的情况下提供宽松的核苷酸选择。本项目的一个重要目标是利用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可以直接与最近的动物SERCA结构进行比较。此外，植物钙泵的细胞分布仍未解决。例如，钙调素刺激的钙泵不仅局限于质膜，“er型”钙泵也不局限于细胞器。因此，另一个目标是确定LCA的膜池，以及它的递送是否通过磷酸化调节。本研究的目标将通过以下方法实现：1)在酵母中表达全长野生型和突变型LCA， 2)在大肠杆菌中过表达和纯化LCA的胞内结构域，以及3)通过转基因番茄植物表达绿色荧光蛋白标记的LCA。更广泛的影响。本项目旨在让来自代表性不足群体（主要是非裔美国人和西班牙裔美国人，但也包括美国原住民和太平洋岛民）的本科生积极参与，为了解植物p型atp酶做出重大贡献。这一目标包括通过开发两学分的本科研究导论课程来加强本科课程，该课程将积极招收选修两门核心二年级课程（即遗传学和细胞生物学）的本科少数民族学生。这门以实验为基础的课程将提供批判性思维和科学发现的机会。学生的参与将由首席研究员和实验室的博士生（也参加学者教育计划）共同指导。除了获得学分外，本课程将成为进入论文驱动型本科研究项目的踏脚石。为了提高学生的参与度，政府在暑期为有兴趣的学生提供资助。最后，本科生参加者将协同参与本系目前的外展工作，将当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对代表性不足的学生的保留，并鼓励未来申请者的多样性。

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.