Identification and Characterization of SNX15, a Novel Sorting Nexin Involved in Protein Trafficking

Abstract

Sorting nexins are a family of phox homology domain containing proteins that are homologous to yeast proteins involved in protein trafficking. We have identified a novel 342-amino acid residue sorting nexin, SNX15, and a 252-amino acid splice variant, SNX15A. Unlike many sorting nexins, a SNX15 ortholog has not been identified in yeast or Caenorhabditis elegans. By Northern blot analysis, SNX15 mRNA is widely expressed. Although predicted to be a soluble protein, both endogenous and overexpressed SNX15 are found on membranes and in the cytosol. The phox homology domain of SNX15 is required for its membrane association and for association with the platelet-derived growth factor receptor. We did not detect association of SNX15 with receptors for epidermal growth factor or insulin. However, overexpression of SNX15 led to a decrease in the processing of insulin and hepatocyte growth factor receptors to their mature subunits. Immunofluorescence studies showed that SNX15 overexpression resulted in mislocalization of furin, the endoprotease responsible for cleavage of insulin and hepatocyte growth factor receptors. Based on our data and the existing findings with yeast orthologs of other sorting nexins, we propose that overexpression of SNX15 disrupts the normal trafficking of proteins from the plasma membrane to recycling endosomes or the trans-Golgi network.Intracellular vesicle traffic in both yeast and mammals requires the function of numerous proteins that mediate multiple processes including cargo selection, vesicle budding, and fusion of vesicles with specific targets (1-3). Sorting nexins (SNXs) 1 are a family of widely expressed proteins believed to be part of the complex molecular machinery required for protein trafficking (4). Mammalian SNXs are homologous to several yeast proteins (e.g. Vps5p, Mvp1p, and Grd19p) for which there is strong genetic evidence demonstrating a role in protein trafficking (5-7). SNX1 was identified using the yeast two-hybrid system by virtue of its ability to bind to the cytoplasmic domain of the epidermal growth factor (EGF) receptor (8). Subsequently, SNX1 was shown to also interact with receptors for insulin, platelet-derived growth factor (PDGF), transferrin, and leptin (4). Furthermore, SNX1 is the mammalian ortholog of Vps5p, a yeast protein that is a component of a multimeric complex (termed the "retromer complex") involved in retrograde transport of proteins from prevacuolar endosomes to the TGN (9). We have previously described three additional sorting nexins: SNX2, SNX3, and SNX4 (4); and 10 additional sorting nexins (SNX5-14) have been deposited in GenBank TM . Like SNX1, both SNX2 and SNX4 associate with various receptors. In addition, SNX1, SNX2, and SNX4 assemble into oligomeric structures (4). All 14 SNX molecules contain a phox homology (PX) domain, a conserved sequence of unknown function first identified in the p40 phox and p47 phox subunits of the NADPH oxidase complex (10). PX domains consist of ϳ100 amino acid residues, and most contain a proline-rich sequence ...

Evidence for a Role of SNX16 in Regulating Traffic between the Early and Later Endosomal Compartments

“…The N-terminal PX domain of SNX2 can mediate self-association as well as association with SNX1, whereas the coiled-coil domain of SNX2 can only mediate self-oligomerization by interaction with its own coiled-coil domain. In addition, SNX1 and SNX2 have been found to associate with other sorting nexins and other proteins and are present in larger complexes containing other retromer components, such as Vps35, Vps29, and Vps26 (22,23,37,43). SNX6 (23) and SNX15 (37) also exhibit self-association mediated by the PX domain.…”

“…Post-nuclear supernatant was obtained by centrifugation of the lysate at 1000 ϫ g for 10 min, and 360 l of the post-nuclear supernatant was loaded on the top of a tube containing 360-l aliquots of 40,37,34,31,28,25,22,19,16,13, and 10% (w/v) sucrose in 20 mM Hepes, pH 7.3, 1 mM EDTA, sequentially overlaid. Following centrifugation at 55,000 rpm in an SW60Ti rotor, 250 l were collected from the bottom of the tube, resolved by SDS-PAGE, transferred to nitrocellulose, and then probed with the indicated antibodies as described below.…”

“…Similarly, SNX6 can associate with itself and with SNX1, SNX2, and SNX4 (23); and SNX15 can also associate with itself, SNX1, and SNX2 and weakly with SNX4 (37). Interestingly, SNX3 has not been found to associate with any other sorting nexins (22,23,36,37). To determine whether SNX16 was able to form homo-and hetero-oligomers, we cotransfected cells with HA-tagged SNX16 and Myc-SNX1, Myc-SNX3, Myc-tagged SNX16, or each of its various deletion mutants (Fig.…”

All Phox Homology (PX) Domains from Saccharomyces cerevisiae Specifically Recognize Phosphatidylinositol 3-Phosphate

“…oligomers (25)(26)(27)(28). The exception to this rule is SNX3 (26,27), which contains little more than just the PX domain and (in both yeast and mammals) lacks the coiled-coil regions proposed to drive sorting nexin oligomerization.…”

SLIC‐1/sorting nexin 20: A novel sorting nexin that directs subcellular distribution of PSGL‐1

“…The sorting nexins are a family of trafficking molecules defined by the presence of a Phox homology (PX) domain (for recent reviews, see [18][19][20]). Originally identified in the p40phox and the p47phox subunit of NADPH oxidase, the *120-amino acid PX domain has been subsequently recognized in a number of proteins including sorting nexins and signaling molecules such as phosphatidylinositol 3-kinase (PI 3-kinase) and cytokine-independent survival kinase (CISK) [21][22][23][24][25][26][27][28][29][30][31][32][33]. The PX domain has also been characterized as a phosphoinositide-binding module that can localize both the host protein and a bound protein to the cell membrane or intracellular vesicles (for reviews, see [20,[34][35][36]).…”