Mutations Altering the Mitochondrial-Cytoplasmic Distribution of Mod5p Implicate the Actin Cytoskeleton and mRNA 3′ Ends and/or Protein Synthesis in Mitochondrial Delivery

Abstract

). A mutant allele of MOD5 encoding a protein (Mod5p-I,KR6) located predominantly in mitochondria was constructed. Mutants defective in delivering Mod5p-I,KR6 to mitochondria were sought by selecting cells with increased cytosolic activity of this protein. Twenty-five mutants defining four complementation groups, mdp1, mdp2, mdp3, and mdp4, were found. They are unable to respire at 34؇C or to grow on glucose medium at 38؇C. Cell fractionation studies showed that mdp1, mdp2, and mdp3 mutants have an altered mitochondrial-cytoplasmic distribution of Mod5p. mdp2 can be suppressed by ACT1, the actin-encoding gene. The actin cytoskeleton organization is also aberrant in mdp2 cells. MDP2 is the same as VRP1 (S. Eukaryotic cells have organelles separated physically from one another by lipid bilayers and the cytosol. Most proteins have a single destination such as the nucleus, mitochondria, peroxisomes, lysosomes, or the cell surface. However, we and others have discovered a class of genes that encode sorting isozymes, enzymes found in more than one cellular compartment (references 9, 10, 13, 18, 22, 34, 49, and 50; see also references cited in references 28 and 80).The Saccharomyces cerevisiae MOD5 and CCA1 genes that encode the tRNA processing enzymes N 6 -(⌬ 2 )-isopentenyl PP i : tRNA isopentenyltransferase (IPP transferase) and ATP (CTP):tRNA nucleotidyltransferase, respectively, are unique in that the sorting isozymes they encode are found in two organelles (mitochondria and nuclei) as well as the cytosol (10, 79, 80). MOD5 codes for two proteins that differ from each other by the presence or absence of an amino-terminal extension (28,69). Translation initiation at the first AUG of the open reading frame (ORF) produces Mod5p-I, which is located in mitochondria and the cytosol; translation initiation at the second AUG at codon 12 gives rise to Mod5p-II, located in the cytosol and nucleus (10).The mechanisms that locate proteins to single subcellular destinations have been intensively investigated. The importance of cis-acting sequences on the proteins is clearly established. Inroads are being made in identifying components on the organelles that serve as receptors and import channels. Cytosolic factors play critical roles (for an example, see review in reference 72), and recent observations suggest that the site of a protein's synthesis can also be an important factor in dictating its final destination (reviewed in reference 77). Despite this general outline of protein location, there is a very incomplete understanding of how the cell manages intracellular traffic. The natural distribution and function of sorting isozymes in multiple cellular compartments should provide a powerful tool to apply new genetic approaches to studies of protein delivery. This is because the distribution of sorting isozymes is balanced between multiple destinations, and the balance can be altered by cis-acting mutations as shown by the phenotypes caused by changing ATGs of MOD5 and CCA1 (10,28,80). The balance should also be altered by mu...

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