Deciphering the logic circuit for Golgi membrane traffic

破译高尔基膜交通的逻辑电路

• 批准号: 10557834
• 负责人: BENJAMIN S GLICK
• 金额: $ 66.79万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10557834
• 关键词: Age; Biomedical Research; Cells; Cellular biology; Clathrin Adaptors; Coat Protein Complex I; Communication; Cytoplasm; Development; Early Endosome; Engineering; Fluorescence Microscopy; Goals; Golgi Apparatus; Image; Individual; Integral Membrane Protein; Kinetics; Knowledge; Link; Logic; Mammalian Cell; Mediating; Membrane Protein Traffic; Molecular; Organelles; Pathway interactions; Play; Process; Productivity; Property; Proteins; Recycling; Role; SNAP receptor; Saccharomyces cerevisiae; Saccharomycetales; Sorting; Structure; System; Transcription Factor AP-1; Vesicle; Visit; Visualization; Work; Yeasts; genome editing; improved; insight; lipid metabolism; mutant; novel; protein distribution; tool; trans-Golgi Network; unpublished works

Membrane traffic in the endomembrane system is well characterized at the level of components, but crucial aspects of the engineering logic of this system remain obscure. Definitions of endomembrane system compartments are often fuzzy, and knowledge of the directionalities and functions of membrane traffic pathways is incomplete. A particularly enigmatic organelle is the Golgi apparatus. Studies of yeast cells indicate that Golgi cisternae are transient, maturing structures, with resident Golgi proteins distributing in a polarized manner across cisternae of different ages. The Golgi recycles components internally and also communicates extensively with other endomembrane system organelles, but the links between membrane traffic and Golgi organization are poorly understood. We propose that the Golgi can be productively viewed as a set of maturing cisternae, with various membrane traffic pathways being switched on and off in an orderly way during cisternal maturation. Our goal is to elucidate these Golgi-associated membrane traffic pathways and to dissect the molecular logic circuit that controls them. We use budding yeasts as an experimental system. The secretory pathway in Saccharomyces cerevisiae has an unusual organization: non-stacked Golgi cisternae are scattered throughout the cytoplasm, and based on our recent work, the trans-Golgi network (TGN) serves as an early endosome. These properties simplify the analysis of individual maturing cisternae by 4D fluorescence microscopy. By determining the kinetic signatures of proteins as they arrive and depart during cisternal maturation in wild-type or mutant cells, we can obtain novel insights. Recent discoveries include: (1) COPI vesicles mediate recycling of early but not late Golgi proteins. (2) The AP-1 clathrin adaptor is restricted in yeast to the TGN. This result, taken together with prior work from other groups, implies that AP-1 mediates intra-Golgi recycling downstream of COPI. (3) As revealed by our development of a regulatable fluorescent secretory cargo that can be visualized in maturing cisternae, AP-1 has an unexpected ability to promote intra-Golgi recycling of this secretory cargo. (4) In unpublished work, AP-1 cooperates with the clathrin adaptor Ent5 to drive two sequential pathways of intra-Golgi recycling. Transmembrane proteins that recycle by the various COPI- or AP-1-dependent pathways become concentrated in different cisternae, thereby creating the polarized distribution of proteins across the Golgi. Our ongoing efforts with S. cerevisiae are aimed at a molecular characterization of these membrane traffic pathways. We plan to assign roles in specific pathways to individual vesicle tethers, SNAREs, and lipid metabolism processes. In addition, we will identify functional connections that coordinate the timing of the different pathways. A newer project employs cultured mammalian cells. We will use imaging and genome editing to revisit three phenomena that are seemingly at odds with the cisternal maturation concept: nonlinear cargo exit from the Golgi, exchange of secretory cargoes between Golgi ribbons, and retention of aberrant proteins in the TGN. Those phenomena can potentially all be explained by a conserved pathway involving AP-1-dependent recycling of secretory cargoes. Our ambition is to achieve a unified understanding of how the secretory pathway operates in both yeast and mammalian cells.

膜系统中的膜流量在组件的水平上具有很好的特征，但关键方面 该系统的工程逻辑仍然晦涩难懂。内膜系统隔室的定义通常是模糊的， 了解膜交通路径的方向性和功能是不完整的。一个特别神秘的 Organelle是高尔基体。对酵母细胞的研究表明，高尔基水库是瞬态的，成熟的结构， 驻留高尔基蛋白以极化方式分布在不同年龄的水库中。高尔基人回收组件 在内部，也与其他内膜系统细胞器进行广泛通信，但是 膜交通和高尔基组织的理解很少。我们建议可以有效地查看高尔基 作为一组成熟的水箱，各种膜交通路径以有序的方式打开和关闭 在脑海中成熟期间。我们的目标是阐明这些高尔基相关的膜交通路径并进行剖析 控制它们的分子逻辑电路。 我们将萌芽的酵母作为实验系统。酿酒酵母中的分泌途径有 不寻常的组织：未堆叠的高尔基水库散布在整个细胞质中，并基于我们最近的工作 Trans-Golgi网络（TGN）是早期内体。这些属性简化了单个成熟的分析 通过4D荧光显微镜进行的储水池。通过确定蛋白质到达并离开时的动力学特征 在野生型或突变细胞中的宿合成熟期间，我们可以获得新颖的见解。最近的发现包括：（1）COPI 囊泡介导早期但晚期高尔基蛋白的回收。 （2）AP-1网旋蛋白适配器在酵母中受到限制 TGN。该结果与其他小组的先前工作一起，意味着AP-1介导了高尔基体内回收 Copi的下游。 （3）正如我们开发可调节的荧光分泌货物所揭示的那样 AP-1在成熟的水库中可视化，具有出乎意料的促进该分泌货物内戈尔基内回收利用的能力。 （4）在未发表的工作中，AP-1与网格蛋白适配器ENT5合作，以驱动高尔基体内的两个顺序途​​径 回收。通过各种COPI-或AP-1依赖性途径回收的跨膜蛋白浓缩 在不同的水箱中，从而在高尔基体中产生了蛋白质的极化分布。 我们与酿酒酵母的持续努力是针对这些膜流量的分子表征 途径。我们计划在各个囊泡系，贪食和脂质代谢中为特定途径中的角色分配角色 过程。此外，我们将确定能够协调不同途径时间的功能连接。 一个较新的项目采用培养的哺乳动物细胞。我们将使用成像和基因组编辑来重新访问三个 现象似乎与李斯纳尔成熟概念不符：非线性货物从高尔基人退出， 在高尔基丝带之间交换分泌货物，并在TGN中保留异常蛋白质。那些现象 可以通过涉及分泌货物的AP-1依赖性回收利用的保守途径来解释。我们的 野心是要对分泌途径在酵母和哺乳动物细胞中的运作方式进行统一的理解。