This award supports a collaborative research program from Dr. Anita K. Hopper, Penn. State Univ. Coll. Medicine and Dr. Nancy C. Martin, Univ. Louisville that will examine how single genes provide proteins to more than one compartment within cells. The discovery that this occurs emerged from observations that mutations affecting cytosolic tRNA modification also affect mitochondrial tRNA. The enzymes making these modifications were called sorting isozymes because they came from a single gene and carried out the same function in multiple cellular compartments. Previous results from these investigators showed that by alternative use of in-frame translation initiation codons the yeast genes TRM1, MOD5 and CCA1 each encode multiple isozymes. For each, the isozyme initiating at the first initiator codon is mitochondrial. However, for Trm1p the amino-terminal extension is not essential for mitochondrial import and for Mod5p the long form is also found in the cytosol. The shorter forms of the proteins can be located in nuclei and/or the cytosol. Earlier results from this collaborative project showed: (1) organelles can share information with the rest of the cell; (2) isozymes can be located in more than one compartment; (3) sorting information resides in additional sequences missing from the eubacterial and archaeal counterparts; (4) compartments may have activity provided by more than one isozyme; (5) mitochondrial targeting information located at amino termini prohibits nuclear import. Despite the advances, important questions remain because the mechanisms that underlie #4 and #5 are still not understood. Aim I is designed to understand how Trm1p can possess both mitochondrial and nuclear targeting information and yet not locate to the nucleus. Possible mechanisms by which commitment to mitochondrial import occurs will be tested using a novel system developed by the investigators that assesses where in a cell a protein destined to mitochondria folds. Experiments will also be performed to learn whether mRNA sorting, cytosolic retention, and/or nuclear export function to prohibit nuclear accumulation of Trm1p-I. Sorting isozymes are not only distributed to different organelles; they have distinct suborganellar locations. For example, the investigators showed that two sorting isozymes have distinct subnuclear locations; Trm1p, is located at the inner nuclear membrane (INM) and Mod5p is located in the nucleoplasm and the nucleolus. Goals of Aim II are to confirm and characterize motif(s) specifying INM location and to identify gene products that tether Trm1p to the INM. Yeast mutants that fail to appropriately locate Trm1p to the INM will be characterized. The studies in Aim II will not only address how sorting isozymes locate to the correct suborganellar locations, but, in addition, will provide information concerning nuclear architecture and nuclear biogenesis - an important area of cell biology not understood for any eukaryote.
