Abstract. When nuclear localization sequences (termed NLS) are placed at the N terminus of cytochrome ca, a mitochondrial inner membrane protein, the resulting hybrid proteins do not assemble into mitochondria when synthesized in the yeast Saccharomyces cerevisiae. Cells lacking mitochondrial cytochrome c~, but expressing the hybrid NLScytochrome c, proteins, are unable to grow on glycerol since the hybrid proteins are associated primarily with the nucleus. A similar hybrid protein with a mutant NLS is transported to and assembled into the mitochondria. To identify proteins that might be involved in recognition of nuclear localization signals, we isolated conditional-lethal mutants (npl, for nuclear protein localization) that missorted NLS-cytochrome c, to the mitochondria, allowing growth on glycerol. The gene corresponding to one complementation group (NPLI) encodes a protein with homology to DnaJ, an Escherichia coli heat shock protein, npU-1 is allelic to sec63, a gene that affects transit of nascent secretory proteins across the endoplasmic reticulum. Rothblatt, J. A., R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman. 1989. J. Cell Biol. 109:2641-2652. The npU mutants reported here also weakly affect translocation of preprocarboxypeptidaseY across the ER membrane. A normally nuclear hybrid protein containing a NLS fused to invertase and a nucleolar protein are not localized to the nucleus in np11/sec63 cells at the nonpermissive temperature. Thus, NPLI/SEC63 may act at a very early common step in localization of proteins to the nucleus and the ER. Alternatively, by affecting ER and nuclear envelope assembly, npll may indirectly alter assembly of proteins into the nucleus.ACH organelle in a eukaryotic cell has a distinct set of proteins that are necessary for its specific function. Certain peptides can act as signals to localize proteins to particular organelles such as the ER, the mitochondria (Verner and Schatz, 1988) and the nucleus (Silver and Hall, 1988). Several proteins have been identified that mediate the recognition of ER-destined proteins and their subsequent translocation across or assembly into the ER membrane (Walter and Blobel, 1980;Meyer et al., 1982;Tajima et al., 1986; Wiedrnan et al., 1987). Receptors have been proposed for mitochondrial signal peptides (Pfaller and Neupert, 1987;Pfanner et al., 1987) and recently a receptor for protein import into chloroplasts has been identified (Pain et al., 1988). By analogy, similar components may exist for localization of proteins to the nucleus.Nuclear localization sequences (NLS)' are stretches of amino acids that are capable of redirecting nonnuclear pro-1. Abbreviations used in this paper: DAPI, diamidinophenylindole; EMS, ethyl methanesulfonate; NLS, nuclear localization sequences; preproCPY, preprocarboxypeptidase Y.teins such as/$-galactosidase to the nucleus. When the first 74 amino acids of the yeast DNA binding protein GAL4 are joined to/~-galactosidase, the result is a fusion protein that is found exclusively in the yeast nucleus as...