Activity and sorting of tRNA nucleotidyltransferase

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Proteins are the molecules of life that are responsible for almost all of the activities occurring in cells. To carry out these activities, proteins must fold up into specific three dimensional shapes which will allow them to interact with the things that they need to carry out their activities (e.g., other proteins or metal ions or small molecules). In addition, the structures of these proteins must allow them to be stable in the cell for an appropriate length of time. Some proteins exist only transiently (e.g., those turning on or off genes under specific conditions) while others need to be very stable (e.g., structural components of the cell). Moreover, as cells are not simply bags of molecules, but are divided into discrete sub-cellular compartments each with specific jobs, the proteins must contain signals that ensure their distribution to the appropriate compartments in the cell where their activities are needed. Ultimately, the function of any protein in a cell is defined by a combination of four factors: localization, activity, structure and stability. The protein must be in the right place (localization) with the right activity, for the right length of time (stability) with the appropriate structure (to interact with accessory factors). We use tRNA nucleotidyltransferase, a protein that is essential for cell life, to explore the interplay of these factors in protein function. This enzyme is responsible for generating functional transfer RNAs (tRNAs) which are needed for protein synthesis. If we lack tRNA nucleotidyltransferase, we lack functional tRNAs, we cannot make proteins and we die. Interestingly, tRNA nucleotidyltransferase is required in all of the compartments in the cell where protein synthesis or tRNA maturation takes place (cytosol, nucleus, mitochondrion and, in plants, plastid). So, this protein must fold up in such a way as to make not only a stable, active protein but also one that can be directed to multiple cellular destinations. To explore how localization, activity, structure and stability interact to define the function of this protein, we use the yeast and plant tRNA nucleotidyltransferases as models. We make specific changes in these proteins and use a combination of biochemical, biophysical, genetic and cell biological techniques to determine how those changes affect localization, activity, structure and stability. Interestingly, we have shown that specific changes may affect more than one of these parameters. For example, we were able to make a change in a plant tRNA nucleotidyltransferase that altered its localization (reduced mitochondrial and increased plastid localization), activity (reduced activity to below the level of detection) and stability (altered thermal denaturation), but not its structure (as measured by circular dichroism and fluorescence spectroscopy). Now we will explore the interplay between localization, activity and stability. We will also use a combination of approaches to identify other proteins in the cell that interact with tRNA nucleotidyltransferase to see how these other proteins may regulate or control tRNA nucleotidyltransferase targeting. These studies will allow us to develop a better understanding of how many factors of protein design combine to ensure accurate function in multiple cellular compartments. With this increased understanding we may be able to better design cells to optimize the delivery of functional proteins or to correct errors in localization.