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. ***
