Dual trafficking of Slit3 to mitochondria and cell surface demonstrates novel localization for Slit protein

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isol ated. Each displays overlapping, but distinct, patterns of expression in th e developing vertebrate central nervous system, implying conservation of fu nction. However, vertebrate Slit genes are also expressed in nonneuronal ti ssues where their cellular locations and functions are unknown. In this stu dy, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Sl it genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within t he mitochondria. This localization was confirmed using immunoelectron micro scopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. How ever, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH2-terminal mitocho ndrial localization signal that can direct a reporter green fluorescent pro tein to the mitochondria. The equivalent region from Slit1 cannot elicit mi tochondrial targeting. We conclude that Slit3 protein is targeted to and lo calized at two distinct sites within epithelial cells: the mitochondria, an d then, in more confluent cells, the cell surface. Targeting to both locati ons is driven by specific NH2-terminal sequences. This is the first examina tion of Slit protein localization in nonneuronal cells, and this study impl ies that Slit3 has potentially unique functions not shared by other Slit pr oteins.