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

Abstract

Drosophila slit is a secreted protein involved in midline patterning. Three vertebrate orthologs of the fly slit gene, Slit1, 2, and 3, have been isolated. Each displays overlapping, but distinct, patterns of expression in the developing vertebrate central nervous system, implying conservation of function. However, vertebrate Slit genes are also expressed in nonneuronal tissues where their cellular locations and functions are unknown. In this study, we characterized the cellular distribution and processing of mammalian Slit3 gene product, the least evolutionarily conserved of the vertebrate Slit genes, in kidney epithelial cells, using both cellular fractionation and immunolabeling. Slit3, but not Slit2, was predominantly localized within the mitochondria. This localization was confirmed using immunoelectron microscopy in cell lines and in mouse kidney proximal tubule cells. In confluent epithelial monolayers, Slit3 was also transported to the cell surface. However, we found no evidence of Slit3 proteolytic processing similar to that seen for Slit2. We demonstrated that Slit3 contains an NH(2)-terminal mitochondrial localization signal that can direct a reporter green fluorescent protein to the mitochondria. The equivalent region from Slit1 cannot elicit mitochondrial targeting. We conclude that Slit3 protein is targeted to and localized at two distinct sites within epithelial cells: the mitochondria, and then, in more confluent cells, the cell surface. Targeting to both locations is driven by specific NH(2)-terminal sequences. This is the first examination of Slit protein localization in nonneuronal cells, and this study implies that Slit3 has potentially unique functions not shared by other Slit proteins.

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

“…Other constructs used in this study were human BMP4 (PMT21-BMP4 myc , a gift of Dr. Tom Jessell), chicken Chordin (pMT21-CHRD HA , a gift of Dr. Kevin Lee), and human growth hormone receptor (pcDNA3-GHR HA , a gift of Prof. Michael Waters). Generation of Anti-CRIM1 Antibodies-Polyclonal antibodies were raised and purified as described previously (26). ␣-NC was raised toward a synthetic peptide: KVCQPGYLNILVSKASGKPGEC (Chiron Technologies).…”

“…␣-NC was raised toward a synthetic peptide: KVCQPGYLNILVSKASGKPGEC (Chiron Technologies). ␣-CC was raised toward a glutathione S-transferaseconjugated C-terminal epitope of mouse CRIM1 (residues 998 -1036; GenBank TM accession number AF168680), which was cloned into pGEX as described previously (26).…”

“…Western blotting, of fractionated samples, cellular extracts, or media as described below, was performed as described previously (26).…”

“…Immunofluorescence of Cells-Immunofluorescence on cell lines using the antibodies indicated was performed as described previously for permeabilized cells (26). For cell surface binding, antibody incubations were performed on live cells incubated on ice to inhibit internalization.…”

The Efficiency of Protein Compartmentalization into the Secretory Pathway

“…More recently, it has been demonstrated to interact with the cytosolic adaptor protein Grb3 during signal transduction (Goh et al, 1996;Kanai et al, 1997), shuttle between the nucleus and cytosol (Forough et al, 2003), and modulate calcium homeostasis by binding to the cytosolic tail of the TRP5 channel (Gkika et al, 2004). Other secretory pathway proteins with alternative localizations include mitochondrial transforming growth factor-␤ (Heine et al, 1991), mitochondrial Slit3 (Little et al, 2001), mitochondrial cytochrome p450 (Addya et al, 1997;Anandatheerthavarada et al, 1999), cytosolic IL15 (Tagaya et al, 1997;Kurys et al, 2000), nucleocytoplasmic cathepsin L (Goulet et al, 2004), and nuclear HBV precore protein (Garcia et al, 1988;Ou et al, 1989).…”