MDP1, A SACCHAROMYCES-CEREVISIAE GENE INVOLVED IN MITOCHONDRIAL CYTOPLASMIC PROTEIN DISTRIBUTION, IS IDENTICAL TO THE UBIQUITIN-PROTEIN LIGASE GENE RSP5/

Alteration of the subcellular distribution of Mod5p-I, a tRNA modifica tion enzyme, member of the sorting isozyme family, affects tRNA-mediat ed nonsense suppression. Altered suppression efficiency was used to id entify MDP genes, which, when mutant, change the mitochondrial/cytosol ic distribution of Mod5p-I,KR6. MDP2 is the previously identified VRP1 , which encodes verprolin, required for proper organization of the act in cytoskeleton. MDP3 is identical to PAN1, which encodes a protein in volved in initiation of translation and actin cytoskeleton organizatio n. We report here the cloning and characterization of wild-type and mu tant MDP1 alleles and the isolation and characterization of a multicop y suppressor of mdp1 mutations. MDP1 is identical to RSP5, which encod es ubiquitin-protein ligase, and mdp1 mutations are suppressed by high copy expression of ubiquitin. All four characterized mdp1 mutations c ause missense changes located in the hect domain of Rsp5p that is high ly conserved among ubiquitin-protein ligases. In addition to its well- known function in protein turnover, ubiquitination has been proposed t o play roles in subcellular sorting of proteins via endocytosis and in delivery of proteins to peroxisomes, the endoplasmic reticulum and mi tochondria. mdp1, as well as mdp2/vrp1 and mdp3/pan1 mutations, affect endocytosis. Further, mdp1 mutations show synthetic interactions with mdp2/vrp1 and mdp3/pan1. Identification of MDP1 as RSP5, along with o ur previous identification of MDP2/VRP1 and MDP3/PAN1, implicate inter actions of the ubiquitin system, the actin cytoskeleton and protein sy nthesis in the subcellular distribution of proteins.