Deciphering the logic circuit for Golgi membrane traffic

内膜系统中的膜运输在成分水平上有很好的特征，但关键的方面是 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， 对膜通道的方向性和功能的了解也是不完整的。一个特别神秘的人 细胞器是高尔基体。对酵母细胞的研究表明，高尔基池是暂时的、成熟的结构，具有 居留高尔基体蛋白以极化的方式分布在不同年龄的脑池中。高尔基回收利用零部件 在内部，也与其他内膜系统细胞器广泛沟通，但之间的联系 膜运输和高尔基体组织还知之甚少。我们认为高尔基可以被富有成效地观看 作为一组成熟的池子，各种膜通道有序地开关 在脑池成熟期间。我们的目标是阐明这些高尔基体相关的膜运输途径，并解剖 控制它们的分子逻辑电路。 我们使用发芽酵母菌作为实验系统。酿酒酵母的分泌途径有一个 不寻常的组织：非堆叠的高尔基体池散布在细胞质中，根据我们最近的工作， 跨高尔基体网络(TGN)是早期的内体。这些特性简化了对个体成熟度的分析 4D荧光显微镜下观察脑池。通过确定蛋白质到达和离开时的运动特征 在野生型或突变细胞的脑池成熟过程中，我们可以获得新的见解。最近的发现包括：(1)COPI 囊泡调节早期高尔基体蛋白质的循环，而不是晚期高尔基体蛋白质。(2)AP-1网状蛋白适配器在酵母中仅限于 TGN.这一结果，与其他小组先前的工作相结合，表明AP-1介导高尔基体内循环 在COPI下游。(3)正如我们开发的可调节的荧光分泌货物所揭示的那样， 在成熟的脑池中可以看到，AP-1具有一种意想不到的能力，可以促进这种分泌货物在高尔基体内的循环。 (4)在未发表的工作中，AP-1与笼状蛋白适配器Ent5合作，驱动高尔基体内的两条连续通路 回收利用。通过依赖COPI或AP-1的各种途径循环的跨膜蛋白变得集中 在不同的池子里，从而造成蛋白质在高尔基体中的极化分布。 我们对酿酒酵母正在进行的努力旨在对这些膜运输进行分子表征。 小路。我们计划将特定路径中的角色分配给单独的囊泡系绳、圈套和脂类代谢。 流程。此外，我们还将确定协调不同通路的时序的功能连接。 一个较新的项目使用了培养的哺乳动物细胞。我们将使用成像和基因组编辑来重温三个 似乎与脑池成熟概念不符的现象：非线性货物从高尔基山脉流出， 高尔基带之间的分泌物质交换，以及异常蛋白在TGN中的保留。那些现象 所有这些都可能被一条保守的途径所解释，该途径涉及AP-1依赖的分泌货物的回收。我们的 其目标是对酵母和哺乳动物细胞中的分泌途径如何运作达成统一的理解。

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.