Structure and Function of Coatomer

这个项目解决了细胞内膜交通的问题；即，细胞如何将膜片段从细胞中的一个物理位置移动到另一个物理位置？虽然在过去的二十年里，关于这一极其复杂的过程已经了解了很多，但仍然有许多问题没有得到回答。这个项目关注的是一种特殊蛋白质复合体的功能。COPI是一种由ADP-核糖化因子1(ARF1)和辅酶原组成的大分子蛋白质复合体，它参与了细胞内某些膜从一个地方到另一个地方的特定运动，以及某些膜蛋白在被移动的膜中的特定包含物。ARF1是一种小的GTP结合蛋白，它将辅助剂招募到高尔基膜上，辅助剂是一种可溶的复合体，包含七个蛋白质亚基α-到Zeta-COP；有一个β和一个β-质数。在高尔基体膜上组装后，COPI包围着从膜上萌发的新生小泡。一旦囊泡形成，ARF1将GTP和COPI从膜上解离，将辅酶和ARF1释放到细胞质中，进行另一轮包衣。虽然已知COPI循环的一般特征，但对COPI作用的功能和分子机制知之甚少。大多数研究人员同意COPI包被的囊泡以逆行的方向携带膜，从高尔基体到内质网(ER)，但对于COPI包被的囊泡是否也以顺行方向运膜，即朝向质膜，存在分歧。目前尚不清楚COPI如何仅与某些膜结合，COPI包裹的囊泡是如何形成的，或者COPI确切地如何帮助选择要包括在萌芽囊泡中的货物。本项目的目的是为了更好地了解COPI的功能和作用。参与该项目的将是一名博士后研究员、一名博士生以及轮流的研究生和本科生。这项工作将在大学环境中完成，在大学环境中，PI既有研究生和本科生的教学责任，也有研究责任。Cotom包含与某些跨膜蛋白胞质区域中的氨基酸基序结合的位点。在选择要包括在COPI涂层囊泡中的货物时，涉及将这些基序与辅酶A结合。最具特性的细胞质基序是双赖氨酸信号(KKXX和相关序列)，它在逃逸到高尔基复合体的常驻ER蛋白返回内质网的过程中发挥作用。在之前NSF拨款支持的已发表工作中，Draper博士研究了似乎模仿双赖氨酸基序并抑制β-COP和Delta-COP与高尔基膜结合的双氨基化合物。该奖项支持的研究是先前工作的延伸，包含两个相关的子项目。子项目1是调查双氨基化合物和多肽引起的辅酶原变化。德雷珀博士最近发现，某些似乎与辅助剂上的双赖氨酸结合部位相互作用的双氨基化合物会将大型辅助剂复合体解离为较小的颗粒。这项工作将继续确定辅助剂是否被作为辅助剂天然配体的多肽解离成更小的颗粒。Cotom将与多肽孵育，并将通过速度沉降离心法分析亚复合体的形成。还将确定亚复合体中有哪些辅酶原亚单位。子项目2是了解双氨基化合物和多肽引起辅酶原基团变化的功能后果。德雷珀博士有初步证据表明，辅助剂在双氨基化合物存在下形成的亚复合体保留了部分功能，这项工作将扩展到研究与辅助剂相互作用的多肽。为了评估功能，高尔基体膜将与胞浆和一种有或没有多肽的能源孵育。哪些辅酶亚基保留高尔基体结合能力将通过免疫印迹来确定。因此，本项目将研究含二赖氨基的多肽在COPI功能中可能发挥的特定作用。如果通过这些和随后的实验证实这种作用，它将对我们对膜运输和特异性的理解产生重大影响。此外，该项目将成为教育下一代科学家以及教育我们社会中有科学素养的成员的工具。

This project addresses the question of intracellular membrane traffic; i.e., how do cells move pieces of membranes from one physical location in the cell to another? While much has been learned about this extremely complicated process over the past twenty years, there are still many questions that remain unanswered. This project focuses on the function of a particular protein complex. COPI, that has been known to be involved in the specific movement of certain membranes from one place to another in the cell and in the specific inclusion of certain membrane proteins in the membranes that are moved.Coat protein I (COPI) is a macromolecular protein complex consisting of ADP-ribosylation factor 1 (ARF1) and coatomer. ARF1 is a small GTP-binding protein that recruits coatomer to Golgi membranes and coatomer is a soluble complex that contains seven protein subunits alpha- through zeta-COP; there's a beta and a beta-prime). After assembly on Golgi membranes, COPI surrounds nascent vesicles that bud from the membrane. Once a vesicle has formed, ARF1 hydrolyzes GTP and COPI dissociates from the membrane, releasing coatomer and ARF1 into the cytosol for another round of coating. Although general features of the COPI cycle are known, the function and molecular mechanism of COPI action are poorly understood. Most researchers agree that COPI-coated vesicles carry membrane in a retrograde direction, from the Golgi to the endoplasmic reticulum (ER), but there is disagreement whether COPI-coated vesicles also carry membrane in the anterograde direction, towards the plasma membrane. It is not understood how COPI binds only to certain membranes, how vesicles coated by COPI form, or precisely how COPI helps select cargo to be included in a budding vesicle. The objective of this project is to better understand the function and action of COPI. Participating in the project will be a post-doctoral fellow, a Ph.D. student, plus rotating graduate and undergraduate students. The work will be done in a university environment where the PI has teaching responsibilities in graduate and undergraduate classes as well as research responsibilities. Coatomer contains sites that bind amino acid motifs present in the cytoplasmic domains of certain transmembrane proteins. Binding of these motifs to coatomer is involved in selecting cargo to be included in a COPI-coated vesicle. The best-characterized cytoplasmic motif is the dilysine signal (KKXX and related sequences) that functions in the return back to the ER of resident ER proteins that have escaped to the Golgi complex. In published work supported by a previous NSF grant, Dr. Draper studied di-amino compounds that appear to mimic the dilysine motif and inhibit the association of beta-COP and delta-COP with Golgi membranes. The research supported by this award is an extension of that previous work and contains two related sub-projects.Sub-project 1 is to investigate changes in coatomer caused by di-amino compounds and peptides. Dr. Draper has recently discovered that certain di-amino compounds that appear to interact with the dilysine binding sites on coatomer dissociate the large coatomer complex into smaller particles. This work will be continued to determine whether coatomer is dissociated into smaller particles by peptides that are natural ligands for coatomer. Coatomer will be incubated with peptides and the formation of sub-complexes will be analyzed by velocity sedimentation centrifugation. Which coatomer subunits are in the sub-complexes will also be determined.Sub-project 2 is to understand the functional consequences of changes in coatomer caused by di-amino compounds and peptides. Dr. Draper has preliminary evidence that sub-complexes formed by coatomer in the presence of di-amino compounds retain partial function, and this work will be extended to study peptides that interact with coatomer. To assess function, Golgi membranes will be incubated with cytosol and an energy source in the presence or absence of peptides. Which coatomer subunits retain Golgi-binding ability will be determined by immunoblotting.This project will thus examine the possible specific role played by the di-lysyl bearing peptides in COPI function. If such a role is substantiated through these and subsequent experiments, it would have a major impact on our understanding of membrane trafficking and specificity. Additionally, the project will serve as a vehicle for the education of the next generation of scientists as well as for the education of scientifically literate members of our society.