Secretory Pathway Calcium and Manganese Pumps

分泌途径钙和锰泵

• 批准号: 7150782
• 负责人: RAJINI RAO
• 金额: $ 31.87万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7150782
• 关键词: Golgi apparatus; bioenergetics; calcium; calcium transporting ATPase; cell membrane; chemical kinetics; computer simulation; conformation; fungal genetics; fungal proteins; intermolecular interaction; intestinal mucosa; ion transport; manganese; molecular site; protein folding; protein purification; protein structure function; radiotracer; scintillation counter; site directed mutagenesis; structural biology; tissue /cell culture

DESCRIPTION (provided by applicant): Secretory Pathway Calcium-ATPases (SPCA) are a newly defined family of ion pumps that transport calcium and manganese into the lumen of the Golgi apparatus where they are essential for sorting, processing, and glycosylation of proteins. The first member of this family, named PMR1, was described in Saccharomyces cerevisae, and more recently, 2 mammalian homologues of PMR1, SPCA1 and SPCA2, have been identified. Mutations in hSPCAl cause Hailey Hailey disease, a debilitating disorder characterized by severe ulceration of the skin, thought to result from dysregulation of cellular calcium. Excess manganese is deposited in the brain and leads to Parkinsonism. The SPCA have been implicated in diverse physiological processes, ranging from manganese detoxification in the liver, calcium transport across intestinal epithelia, and milk production by the mammary glands, although there is little molecular evidence for their specific roles. This proposal combines 3 parallel approaches to investigate the SPCA: biochemical studies using purified proteins or Golgi membranes, cell biological studies in polarized cultured mammalian cells, and large scale phenomic analysis of ion homeostasis in a model organism. In previous studies, we have defined the transmembrane helices and residues critical for ion transport and have identified a role for helix packing in determining ion selectivity. In Aim 1of this proposal, we will shift our focus to understanding the ion binding and modulatory role of EF motifs in the cytoplasmic N-terminal domain. We have new evidence for trafficking of the pumps between the Golgi stacks and a novel, vesicular compartment in polarized cell models of hepatocytes and enterocytes. In Aim 2, we will determine if trafficking is ion-dependent and related to transcellular transport, and whether specific retrieval or PDZ-binding motifs at the C-terminus are important for localization. Gene knockdown approaches will be used to evaluate the isoform-specific contributions of the 2 SPCA pumps in the enterocyte model. Finally, we propose to use the yeast model organism for high resolution phenomic analysis that will identify new genes and pathways associated with the cellular function of the SPCA pumps (Aim 3). Taken together, this proposal lays the foundation for understanding the role of this novel family of transporters at the molecular, cellular and physiological level.

描述（由申请人提供）：分泌途径钙-ATPase（SPCA）是一个新定义的离子泵家族，将钙和锰将钙和锰转运到高尔基体的腔内，对于蛋白质的分类，加工和糖基是必不可少的。该家族的第一个成员，名为PMR1的成员，在酿酒酵母中描述了，最近已经确定了2个PMR1，SPCA1和SPCA2的哺乳动物同源物。 HSPCAL的突变引起Hailey Hailey病，这是一种具有严重溃疡皮肤的衰弱性疾病，被认为是由于细胞钙的失调而引起的。多余的锰被沉积在大脑中，并导致帕金森氏症。 SPCA与多种生理过程有关，范围从肝脏中的锰排毒，跨肠道上皮的钙转运以及乳腺产生牛奶，尽管几乎没有分子证据证明其特定角色。该提议结合了研究SPCA的3种平行方法：使用纯化的蛋白质或高尔基膜，极化培养的哺乳动物细胞中的细胞生物学研究以及模型有机体中离子稳态的大规模现象分析。在先前的研究中，我们定义了跨膜螺旋和离子转运至关重要的残基，并确定了螺旋填料在确定离子选择性中的作用。在该提案的目标1中，我们将把重点转移到理解EF基序在细胞质N末端结构域中的离子结合和调节作用。我们有新的证据表明高尔基堆积之间的泵和肝细胞和肠西细胞极化细胞模型中的新颖的水泡区室。在AIM 2中，我们将确定运输是否依赖于离子依赖性并且与跨细胞运输有关，以及C-terminus的特定检索或PDZ结合基序是否对定位很重要。基因敲低方法将用于评估肠道细胞模型中2个SPCA泵的同工型特异性贡献。最后，我们建议将酵母模型生物用于高分辨率现象分析，该分析将识别与SPCA泵的细胞功能相关的新基因和途径（AIM 3）。综上所述，该建议为理解这种新型转运蛋白家族在分子，细胞和生理水平上的作用奠定了基础。