Protein Transport Across Membranes

蛋白质跨膜运输

基本信息

批准号：
10655014
负责人：
Tom A Rapoport
金额：
$ 52.38万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-05-01 至 2027-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10655014
关键词：
26S proteasome ATP phosphohydrolase Address Anabolism Back Binding Biogenesis Biological Assay C-terminal Cells Complex Cryoelectron Microscopy Cystic Fibrosis Cytosol Disease Docking Endoplasmic Reticulum Endoplasmic Reticulum Degradation Pathway Event Funding Genetic Diseases Glycoproteins Glycoside Hydrolases Goals Homo Human Genetics Ligase Lipid Binding Mammals Mediating Medical Membrane Membrane Proteins Metabolism Modeling Monoubiquitination Nuclear Pore Organelles Paper Pathway interactions Peroxisomal Disorders Process Progress Reports Protein Import Protein translocation Proteins Publications Publishing Recycling Saccharomyces cerevisiae Signal Transduction Structure System Testing Thinness Toxin Ubiquitination Virus Work Xenopus Zellweger Syndrome alpha 1-Antitrypsin Deficiency aqueous cofactor crosslink design dimer drug development egg experimental study misfolded protein multicatalytic endopeptidase complex novel novel therapeutics peroxisome peroxisome lumen protein degradation protein oligomer protein purification protein transport receptor receptor binding receptor recycling reconstitution ubiquitin ligase

项目摘要

PROJECT SUMMARY The goal of this project is to understand in mechanistic terms how proteins are transported across membranes. One aspect of this proposal is to clarify how misfolded endoplasmic reticulum (ER) proteins are retro-translocated from the ER into the cytosol (ER-associated protein degradation, ERAD). The proposal is based on recently determined cryo-electron microscopy (cryoEM) structures of the Hrd1 complex that elucidated how luminal proteins are retro-translocated, and on experiments that clarified how the Cdc48 ATPase processes its substrates. Another aspect of the proposal deals with protein import into peroxisomes. Our recent results indicate that folded cargo translocates with its receptor into the lumen of peroxisomes; the receptor subsequently returns to the cytosol through a retro-translocon formed by a heterotrimeric ubiquitin ligase complex. Here, we will address central outstanding questions of these protein translocation pathways: Specific aim #1: How are proteins moved from the ER lumen across the membrane? We will clarify the mechanism by which misfolded glycoproteins are delivered to the Hrd1 complex and moved through the membrane. Specific aim #2: How are substrates moved from the ER membrane to the proteasome? We propose to determine how the Cdc48 complex moves substrates into the cytosol and test the hypothesis that membrane proteins are extracted together with bound lipids. We will investigate how shuttling factors and Cdc48- binding cofactors transfer substrates between Cdc48 and the 26S proteasome. Specific aim #3: How are proteins imported into peroxisomes? Based on preliminary experiments, we propose to test the hypothesis that Pex13 forms a nuclear pore-like structure through which cargo-bound receptors move across the peroxisomal membrane. Specific aim #4: How are receptors recycled back into the cytosol? We will test our model that the ubiquitin ligase complex forms a retro-translocation channel for recycling receptors. We will use purified proteins to reconstitute ubiquitination by the ligase complex and the subsequent membrane extraction by the Pex1/Pex6 ATPase. The mechanism of ERAD and of peroxisomal protein import are of great medical importance. Many diseases, including cystic fibrosis, are caused by the misfolding of ER proteins and their degradation. The pathway is also hijacked by certain viruses and toxins, and a better understanding may lead to new drugs allowing interference. Our work may also lead to a better understanding and treatment of peroxisomal disorders, such as Zellweger syndrome.

项目概要该项目的目标是从机械角度理解蛋白质如何转运膜。该提案的一个方面是阐明错误折叠的内质网 (ER) 蛋白是如何发生的从 ER 逆转录至细胞质（ER 相关蛋白降解，ERAD）。该提案是基于最近确定的 Hrd1 复合物的冷冻电子显微镜 (cryoEM) 结构，该结构阐明了管腔蛋白如何逆向转位，以及阐明 Cdc48 ATPase 如何处理的实验它的基材。该提案的另一个方面涉及蛋白质导入过氧化物酶体。我们最近的结果表明折叠的货物与其受体一起易位到过氧化物酶体的内腔中；随后受体通过异三聚体泛素连接酶复合物形成的逆转录转位子返回胞质溶胶。在这里，我们将解决这些蛋白质易位途径的核心突出问题：具体目标#1：蛋白质如何从内质网腔穿过膜？我们将阐明错误折叠的糖蛋白被传递至 Hrd1 复合物并移动的机制透过膜。具体目标#2：底物如何从内质网膜转移到蛋白酶体？我们建议确定 Cdc48 复合物如何将底物移动到细胞质中，并检验以下假设：膜蛋白与结合的脂质一起被提取。我们将研究穿梭因子和 Cdc48- 结合辅因子在 Cdc48 和 26S 蛋白酶体之间转移底物。具体目标#3：蛋白质如何导入过氧化物酶体？基于初步实验，我们建议检验 Pex13 形成核孔状的假设货物结合受体穿过过氧化物酶体膜的结构。具体目标#4：受体如何循环回到细胞质中？我们将测试我们的模型，即泛素连接酶复合物形成回收的逆向易位通道受体。我们将使用纯化的蛋白质通过连接酶复合物和随后的泛素化来重建通过 Pex1/Pex6 ATP 酶进行膜提取。 ERAD 和过氧化物酶体蛋白输入的机制具有重要的医学意义。许多疾病，包括囊性纤维化，是由 ER 蛋白的错误折叠及其降解引起的。途径也是被某些病毒和毒素劫持，更好的理解可能会导致新的药物允许干扰。我们的工作还可能有助于更好地理解和治疗过氧化物酶体疾病，例如齐薇格综合症。

项目成果

期刊论文数量（63）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Preserving the membrane barrier for small molecules during bacterial protein translocation.

在细菌蛋白质易位过程中保护小分子的膜屏障。

DOI：
发表时间：
2011-05-12
期刊：
影响因子：
64.8
作者：
Park, Eunyong;Rapoport, Tom A
通讯作者：
Rapoport, Tom A

Key steps in ERAD of luminal ER proteins reconstituted with purified components.

用纯化组分重构管腔 ER 蛋白的 ERAD 关键步骤。

DOI：
发表时间：
2014-09-11
期刊：
影响因子：
64.5
作者：
Stein, Alexander;Ruggiano, Annamaria;Carvalho, Pedro;Rapoport, Tom A
通讯作者：
Rapoport, Tom A

Structures of the double-ring AAA ATPase Pex1-Pex6 involved in peroxisome biogenesis.

参与过氧化物酶体生物合成的双环 AAA ATP 酶 Pex1-Pex6 的结构。

DOI：
发表时间：
2016-03
期刊：
影响因子：
0
作者：
Tan, Dongyan;Blok, Neil B;Rapoport, Tom A;Walz, Thomas
通讯作者：
Walz, Thomas

Two alternative binding mechanisms connect the protein translocation Sec71-Sec72 complex with heat shock proteins.

两种替代的结合机制将蛋白质易位 Sec71-Sec72 复合物与热休克蛋白连接起来。

DOI：
发表时间：
2017
期刊：
影响因子：
0
作者：
Tripathi, Arati;Mandon, Elisabet C;Gilmore, Reid;Rapoport, Tom A
通讯作者：
Rapoport, Tom A

Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1-Npl4.

Cdc48 ATPase 及其泛素结合辅因子 Ufd1-Npl4 的结构。

DOI：
发表时间：
2018
期刊：
影响因子：
16.8
作者：
Bodnar, Nicholas O;Kim, Kelly H;Ji, Zhejian;Wales, Thomas E;Svetlov, Vladimir;Nudler, Evgeny;Engen, John R;Walz, Thomas;Rapoport, Tom A
通讯作者：
Rapoport, Tom A