How Single Genes Provide Proteins to Multiple Cellular Compartments; Collaborative Research

[506810]霍珀和马丁霍珀和马丁实验室合作研究一种不同寻常的酵母基因，这种基因编码“分选同工酶”。这些酶由相同的基因编码，但位于多个亚细胞区室。对这些基因的研究提供了将蛋白质靶向到正确目的地的顺式信号的信息，并有望提供一种揭示在分选过程中发挥作用的基因产物的方法。TRM1编码2种同工酶，一种在线粒体中发现的氨基末端延伸蛋白和一种在线粒体和细胞核中发现的缺乏延伸蛋白，MOD5也编码2种同工酶；氨基端延伸的Mod5p在线粒体中，缺乏这种延伸的形式在细胞核中，但细胞质有两种同工酶的贡献。CCA1编码3个同工酶。氨基末端延长最长的形式位于线粒体中，其他两种形式位于细胞质/核室中。Trm1p、Mod5p、Cca1p均参与tRNA的生物合成。核基因组编码的trna位于细胞质和细胞核中。拟议研究的一个目标是确定细胞是否需要细胞核和细胞质形式的Mod5p和Cca1p来进行反应。在解决这个问题时，将确定这些蛋白质的核输入所必需和充分的序列。然后，这些序列将被改变，以确定错误定位的后果。Trm1p的氨基延伸形式，可能还有Cca1和Mod5p，同时具有线粒体和细胞核的靶向信息，但在细胞核中没有发现。我们将探索能够解释线粒体靶向信息占主导地位的机制。特别是，线粒体靶向信号在耦合输入到翻译中起作用的假设将被测试。这些研究是及时的，因为新的信息表明翻译和线粒体蛋白输入有关。真核细胞的正常功能依赖于蛋白质的适当亚细胞分布。尽管有越来越多的文献讨论了确保适当分发的交互作用组件，但仍有许多东西有待学习。PI的假设是，分选同工酶在多个细胞区室中的自然分布可以作为研究蛋白质向线粒体和细胞核传递的强大新工具，并且这些研究的结果可能对理解具有单个亚细胞目的地和具有多个目的地的蛋白质的分选具有普遍适用性。对排序同工酶分布所必需的序列的理解使PI能够设计遗传研究来确定影响分布过程的成分。首次使用Mod5p进行的基因研究鉴定出4个基因：MDP1/RSP5、MDP2/VRP1、MDP4和PAN1。MDP2/VRP1和PAN1突变导致Mod5p线粒体池减少。其他线粒体蛋白也可能受到影响，因为这些突变会导致呼吸缺陷和条件生长，尽管Mod5p本身并不重要。MDP2/影响肌动蛋白细胞骨架，PAN1编码一种与mRNA 3'端相互作用并影响蛋白质合成起始的蛋白质。发现MDP2 PAN1牵连到肌动蛋白细胞骨架、蛋白质合成和3 '信使rna序列分布的线粒体和细胞质之间的蛋白质。提出了对MDP2和PAN1作用机制的研究。使用一种分类同工酶成功识别出改变其分布到线粒体的基因，预示着补充研究成功识别出改变Mod5p和Cca1p分布的其他基因。在提议的工作中描述了实现这一目标的遗传方案。这是一个由两位女科学家合作的项目，一位是生化学家，一位是遗传学家，她们使用多学科方法研究生物技术中的一个基本问题：单个基因产物的蛋白质如何在细胞内的多个位置进行分类。细胞蛋白质运输背后的基本问题是如何在结构复杂的细胞质中协调蛋白质运输，细胞质由许多不同的膜结合和非膜分隔的区室组成。如果一种蛋白质被靶向到一个单独的隔室，它最初可以用一个“靶向信号”来制造，这个信号随后可以被切割，这取决于它对蛋白质的后续功能是否必要。线粒体和细胞核的靶向信号已知有几种蛋白质只存在于这些隔室中，但本项目涉及一些参与tRNA修饰的蛋白质，这些蛋白质在线粒体和细胞核中都发现，并且包含两种类型的信号。该项目研究了当这些信号以改变线粒体和细胞核之间蛋白质平衡的方式改变时会发生什么。虽然这种蛋白质是由单一基因编码的，但存在不同大小的蛋白质，较长的蛋白质存在于线粒体中，较短的蛋白质存在于细胞核中。基因分析发现了一些有趣的基因，这些基因可以改变线粒体中较长形式的数量。其中一个影响蛋白质合成的起始，另一个影响细胞的肌动蛋白骨架。研究了蛋白质的合成（翻译）与线粒体靶向的偶联。该项目还研究了如何改变蛋白质合成的起始和肌动蛋白细胞骨架可以改变蛋白质在细胞核和线粒体之间的分布。这项研究提供了对维持复杂细胞区隔所必需的分类信息是如何通过新生成的蛋白质与其他细胞成分的相互作用来调节的见解。* * *

9506810 Hopper and Martin The Hopper and Martin laboratories collaboratively study an unusual category of yeast genes that code for "sorting isozymes". These are enzymes coded by the same gene but located in more than one subcellular compartment. Study of these genes has provided information about the cis signals that target proteins to their correct destination, and promises to provide a means of uncovering gene products that play roles in the sorting process. TRM1 codes for 2 isozymes, an amino-terminal extended protein found in mitochondria and a protein lacking the extension found in mitochondria and nuclei, MOD5 also encodes 2 isozymes; the amino-terminal extended Mod5p is in mitochondria and the form lacking this extension is in nuclei, but the cytosol has contributions from both isozymes. CCA1 codes for 3 isozymes. The amino-terminal extended longest form is located in mitochondria and the other two forms are in cytosol/nuclear compartments. Trm1p, Mod5p, and Cca1p are all involved in tRNA biosynthesis. tRNAs encoded by the nuclear genome are located in the cytosol and the nucleus. One goal of the proposed studies is to determine whether cells require both nuclear and cytoplasmic forms of Mod5p and Cca1p for the reactions they carry out. In addressing this problem, sequences necessary and sufficient for nuclear import of these proteins will be identified. Then the sequences will be altered to determine the consequences of mislocalizing them. The amino-extended forms of Trm1p and probably Cca1 and Mod5p possess both mitochondrial and nuclear targeting information, yet are not found in the nucleus. Mechanisms that could account for the dominance of the mitochondrial targeting information will be explored. In particular, the hypotheses that mitochondrial targeting signals play a role in coupling import to translation will be tested. These studies are timely as new information suggests an association of translation and mitochondrial protein i mport. Proper function of a eukaryotic cell is dependent upon appropriate subcellular distribution of proteins. Although there is a growing body of literature that addresses the trans-acting components that assure appropriate distribution, much remains to be learned. The hypothesis of the PI's is that the natural distribution of sorting isozymes to multiple cellular compartments can be used as a powerful new tool to study protein delivery to mitochondria and nuclei and that the outcome of such studies may have general applicability for understanding the sorting of proteins with a single subcellular destination as well as those with multiple destinations. The understanding of sequences necessary for the distribution of sorting isozymes allowed the PI's to devise genetic studies to identify components that affect the distribution process. The first genetic studies using Mod5p identified 4 genes: MDP1/RSP5, MDP2/VRP1, MDP4, and PAN1. Mutations of MDP2/VRP1 and PAN1 cause a decrease in the mitochondrial pool of Mod5p. Other mitochondrial proteins also may be affected because these mutations cause respiratory deficiency and conditional growth even though Mod5p itself is unessential. MDP2/ affects the actin cytoskeleton and PAN1 codes for a protein that interacts with 3' ends of mRNA and affects initiation of protein synthesis. Finding MDP2 and PAN1 implicates the actin cytoskeleton, proteins synthesis and 3' mRNA sequences in the distribution of proteins between the mitochondrial and the cytoplasm. Studies to decipher the mechanism of action of MDP2 and PAN1 are proposed. Success using one sorting isozyme to identify genes that alter its distribution to mitochondria predicts success of complementary studies to identify additional genes that alter the distribution of Mod5p as well as Cca1p. Genetic schemes to accomplish this are described in the proposed work. %%% This is a project engaged by two collaborating women scientists, one a biochemist and one a geneticist, who use multidisciplinary approaches to a fundamental question in biotechnology: how a protein which is the product of a single gene can be sorted to multiple locations within the cell. The basic problem behind cellular protein traffic is how to coordinate that protein traffic in the context of a structurally complex cytoplasm, composed of many different membrane-bound and non-membrane delimited compartments. If a protein is targeted to a single compartment, it can be initially made with a "targeting signal" which can subsequently be cleaved, depending on whether it is necessary for the protein's subsequent function. Targeting signals for the mitochondria and nucleus are known for several proteins which come to reside solely in those compartments, but this project concerns some proteins which are involved in tRNA modification which are found in both the mitochondria and nucleus and which contain both types of signals. The project examines what happens when these signals are changed in a way which changes the balance of the protein between the mitochondria and nucleus. Although the protein is coded by a single gene, different sizes of the protein exist, with the longer form found in mitochondria and the shorter form in the nucleus. Genetic analysis has found some intriguing genes which alter the amount of the longer form in the mitochondria. One of them affects the initiation of protein synthesis and one affects the actin cytoskeleton of the cell. The coupling of the synthesis (translation) of the protein to targeting to the mitochondria is examined. This project also addresses how altering the initiation of protein synthesis and the actin cytoskeleton can change the distribution of the protein between the nucleus and mitochondrion. This study provides insight into how the sorting information necessary for maintaining complex cellular compartmentation is regulated by the interactions of newly-made proteins with other cellular components. ***