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genome editing; improved; insight; lipid metabolism; mutant; novel; protein distribution; tool; trans-Golgi Network; unpublished works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.

内膜系统中的膜运输在组分水平上得到了很好的表征，但 这个系统的工程逻辑仍然模糊不清。内膜系统隔室的定义通常是模糊的， 并且对膜交通路径的方向性和功能的知识是不完整的。一个特别神秘的 细胞器是高尔基体。对酵母细胞的研究表明，高尔基体池是短暂的成熟结构， 在不同年龄的池中以极化方式分布的常驻高尔基体蛋白。高尔基体成分 内部也广泛地与其他内膜系统细胞器沟通，但之间的联系 膜运输和高尔基体组织知之甚少。我们建议，高尔基体可以被有效地认为是 作为一组成熟的池，各种膜交通途径有序地打开和关闭 脑池成熟期。我们的目标是阐明这些高尔基体相关的膜交通途径，并解剖 控制它们的分子逻辑电路 我们使用芽殖酵母作为实验系统。酿酒酵母中的分泌途径具有 不寻常的组织：非堆叠的高尔基体池分散在整个细胞质中，根据我们最近的工作， 反式高尔基体网络（trans-Golgi network，TGN）作为早期内体。这些特性简化了个体成熟度的分析 通过4D荧光显微镜观察脑池。通过确定蛋白质到达和离开时的动力学特征 在野生型或突变细胞的池成熟过程中，我们可以获得新的见解。最近的发现包括：（1）COPI 囊泡介导早期而非晚期高尔基体蛋白的再循环。(2)AP-1网格蛋白衔接子在酵母中被限制为 TGN。这一结果，与其他小组先前的工作一起，意味着AP-1介导了高尔基体内的再循环 COPI的下游(3)正如我们开发的一种可调节的荧光分泌货物所揭示的那样， 在成熟的脑池中可见，AP-1具有意想不到的促进这种分泌货物的高尔基体内再循环的能力。 (4)在未发表的工作中，AP-1与网格蛋白衔接子Ent 5合作，驱动高尔基体内的两个连续途径， 回收利用。通过各种COPI或AP-1依赖性途径再循环的跨膜蛋白质变得浓缩 在不同的池，从而创造两极分布的蛋白质跨越高尔基体。 我们正在努力与S。酿酒酵母的目的是这些膜交通的分子表征 路径。我们计划将特定通路中的作用分配给单个囊泡栓系、SNARE和脂质代谢 流程.此外，我们将确定协调不同途径的时间的功能连接。 一个较新的项目使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审视三个 似乎与脑池成熟概念不一致的现象：非线性货物从高尔基体排出， 高尔基带之间的分泌物交换，以及异常蛋白质在TGN中的保留。这些现象 可能都可以解释为一个保守的途径，涉及AP-1依赖性回收的分泌货物。我们 我们的目标是对酵母和哺乳动物细胞中分泌途径的作用有一个统一的认识。