R egulated targeting of proteins to appropriate sites of action within cells is accomplished in part by enzymatic modification of the address site that is recognized by modular protein domains. Phox (PX) domains are Ϸ120-residue protein modules initially recognized by sequence homology in NADPH oxidase subunits, sorting nexins, and PI 3-kinases (1). Proteins containing PX domains are associated with specific membrane compartments, and the PX domains of Vam7p, p40 Phox , p47 Phox , CISK, and sorting nexin (SNX) 3 recognize specific phosphatidylinositol (PtdIns) (2-8). All PX domains of yeast are reported to recognize PtdIns (3) P (9). Binding affinities of the yeast proteins, however, differ up to 10 3 -fold in vitro, suggesting that membrane association in vivo depends on additional protein⅐protein interactions (9). SNX1, initially identified via its interaction with epidermal growth factor receptor (EGFR), contains a PX domain in its NH 2 terminus and three predicted coiled-coils in its COOH terminus (10). Other SNX family members also contain coiledcoils (SNX2 and SNX4), contain little sequence beyond a PX domain (SNX3), or contain various functional domains (an RGS domain in SNX13͞RGS-PX1, an SH3 domain in SNX9) (11)(12)(13)(14). hSNX1 interacts with orthologs of three yeast proteins that together with Vp5p, the yeast ortholog of SNX1, form the retromer complex, suggesting conservation of function in vesicular trafficking in the endocytic system (15, 16).We have analyzed the role of the PX and coiled-coil domains of SNX1 in subcellular localization to tubulovesicular structures and in interactions with SNX family members. We assessed the effects of regulated overexpression of the NH 2 terminus of SNX1 that contains the PX domain on ligand-induced down-regulation of EGFR. The results of these studies indicate that both an intact PX domain that recognizes PtdIns lipids and the coiled-coil domains of SNX1 involved in protein⅐protein interactions are required for proper vesicle membrane targeting. Neither domain alone is sufficient. We conclude that SNX1 functions to enhance EGFR trafficking in the endosome to lysosome pathway. Materials and MethodsRabbits were immunized with the purified PX domain of SNX1 (aa 142-269) and the resultant antisera that was affinity purified (8501) specifically recognized SNX1 but not SNX2 or SNX3. The SEFIGA rabbit polyclonal antibody was generated using a peptide corresponding to the COOH terminus of hEGFR.Protein Expression, Purification, and Characterization. Sequences encoding PX domains were amplified in PCR reactions from cDNAs encoding the indicated proteins, digested using appropriate restriction enzymes and cloned into expression vectors. Mutations were prepared using oligonucleotide-directed mutagenesis and verified by sequencing. Proteins were expressed in Escherichia coli and soluble proteins were purified on the appropriate affinity columns (NTA, Glutathione, or Chitin agarose) and chromatographed on a Superdex S200 column. Crosslinking of the SNX1 PX domain was carrie...

No abstract

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1-containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes. INTRODUCTIONThe sorting nexin (SNX) family of proteins, with Ͼ25 members in the human genome, is characterized by sequence-related phox homology (PX) domains that bind membrane-localized phosphatidylinositols (PIs) that are phosphorylated on the inositol ring (Sato et al., 2001;Teasdale et al., 2001;Worby and Dixon, 2002). These PX domains function in targeting SNX proteins to specific vesicular membranes. The descriptor "sorting nexin" was applied to the first discovered member, SNX1, based on the hypothesis that these proteins functioned in sorting vesicular cargo in the endosomal system (Kurten et al., 1996). SNX proteins also contain a variety of other functional domains, including coiled coils, regulator of G protein signaling, Src homology 3, and band four point one, ezrin, radixin, moesin homology domains (Worby and Dixon, 2002). Specific SNX family members are reported to bind various cargo proteins, including the tyrosine kinase receptors for epidermal growth factor, platelet-derived growth factor, and insulin (Kurten et al., 1996;Haft et al., 1998); the protease-activated G protein-coupled receptor (Wang et al., 2002); cargo receptors, including the low-density lipoprotein receptor and the transferrin receptor (Haft et al., 1998;Burden et al., 2004); Ser/Thr kinase receptors of the transforming growth factor-␤ family (Parks et al., 2001); leptin receptors (Haft et al., 1998); P-selectin (Florian et al., 2001); Down's syndrome cell adhesion molecule (Worby et al., 2001); enterophilin (Pons et al., 2003); and the Fanconi anemia complementation group A protein (Otsuki et al., 1999).Evidence that these PX domain-containing proteins function as sorting nexins derives from genetic analysis in Saccharomyces cerevisiae where Vps5, the ortholog of mammalian SNX1 and SNX2, functions in a molecular complex, "the retromer," that recycles Vps10p from the vacuole back to the trans-Golgi (Seaman et al., 1998). Grd19p, the ortholog of mammali...

No abstract