Plasma Membrane Targeting of the Yck2 Casein Kinase 1 Isoform

Yck-1和Yck-2蛋白激酶是酿酒酵母生存所必需的。这两个蛋白是一个在真核细胞中高度保守的丰富的蛋白激酶家族的成员，其性质已经得到了很好的研究，但在大多数生物体中其功能尚不清楚。质膜相关的YCK蛋白激酶是酵母细胞形态发生和胞质分裂所必需的，在细胞周期中富含在细胞膜内的特定位置。通过将绿色荧光蛋白融合到Yck 2p的研究，可以确定Yck 2p到质膜的传递途径需要一个功能正常的分泌途径。这是意想不到的，因为YCK蛋白携带膜定位所必需的C-末端序列，该序列可以直接被Prenyl Transfer酶修饰，而到目前为止，该酶只被已知的Rab家族的ras样GTP酶成员的Prenylate修饰。这项研究旨在通过确定Yck 2p上的顺式作用元件和修饰和定位所需的反式作用因子来了解Yck 2蛋白的靶向机制。此外，预烯基化是否对靶向机制或蛋白质与靶标的相互作用具有功能后果的问题也将得到测试。最后，我们将探索Yck 2激酶在质膜上和质膜外与囊泡结构相关的循环的证据，以表征表面的循环，并确定这是否是Yck在酵母中必不可少的功能的组成部分。这些研究的结果不仅对理解必要的蛋白激酶的定位机制具有重要意义，而且将解决蛋白质预烯基化领域的重要问题--特别是预烯基化是否可以决定这些蛋白质对特定膜的靶向以及这种修饰的功能后果。该项目解决了细胞生物学中的一个基本问题，即特定的蛋白质如何到达它们需要的位置，这些蛋白质需要位于细胞内的特定位置才能发挥其功能？这个项目中研究的蛋白质Yck是一种蛋白激酶，是一种在细胞内信号转导事件中发挥作用的蛋白质，通过磷酸化其他特定蛋白质来激活或失活它们。该项目将探索蛋白质预烯基化在蛋白质亚细胞定位中的作用。预烯基化的生物化学(一种重要的蛋白质翻译后修饰，其结果是特定的脂肪酰基与蛋白质共价加成；这种修饰允许原本不与膜相互作用的蛋白质与膜相互作用)。然而，预烯基化的特定细胞生物学后果还没有被很好地理解。这项研究将在一种模式生物--酿酒酵母中进行，在历史上，它为细胞生物学家提供了大量关于细胞过程的基本信息，这些信息适用于广泛的其他类型的生物，包括植物和哺乳动物。

The Yck1 and Yck2 protein kinases are together essential for viability in Saccharomyces cerevisiae. The two proteins are members of an abundant protein kinase family that is highly conserved in eukaryotic cells, whose properties have been well-studied but whose functions are not yet clear in most organisms. The plasma membrane-associated Yck protein kinases are required for cellular morphogenesis and cytokinesis in yeast, and are enriched at specific sites within the membrane during the cell cycle. Studies with a green fluorescent protein fusion to Yck2p allowed determination that the route of delivery of Yck2p to the plasma membrane requires a functioning secretory pathway. This was unexpected because the Yck proteins carry a C-terminal sequence necessary for membrane localization that could direct modification by a prenyltransferase that heretofore was only known to prenylate members of the Rab family of ras-like GTPases. This research is directed toward understanding the targeting mechanisms of the Yck2 protein by determining the cis-acting elements on Yck2p and the trans-acting factors that are required for modification and localization. Also, the question of whether prenylation has functional consequences, either to the targeting mechanism or to the protein's interaction with targets, will be tested. Finally, evidence that the Yck2 kinase cycles on and off the plasma membrane in association with vesicular structures will be explored, to characterize the apparent cycling and to determine whether this is an integral part of the essential function of Yck in yeast. The results of these studies will be important not only in understanding the localization mechanism of an essential protein kinase, but will address important questions in the area of protein prenylation -- especially the question of whether prenylation can determine targeting of these proteins to particular membranes and the functional consequences of this modification. The project addresses a fundamental question in cell biology, namely, how do specific proteins, that need to be located in specific places inside the cell in order to perform their function, get to where they need to be? The protein under investigation in this project, Yck, is a protein kinase, a type of protein that functions in intracellular signal transduction events by activating or inactivating other specific proteins by phosphorylating them. The project will explore the role of protein prenylation in subcellular localization of proteins. The biochemistry of prenylation (an important post-translational modification of a protein that results from the covalent addition of a specific fatty acyl group to the protein; such a modification allows a protein, that would otherwise not interact with membranes, to interact with membranes) is reasonably well understood. However, the specific cell biological consequences of prenylation are not so well understood. The study will be done in a model organism, the yeast Saccharomyces cerevisiae, that historically has richly provided cell biologists with fundamental information about cellular processes that are applicable to a broad range of other types of organisms, including plants and mammals.