Fig. 1. Identification and cellular distribution of SNX16. (A) Domain structure of SNX16. (B) Multiple sequence alignment of sorting nexins (SNXs) 1-4 (GenBank accession nos. U53225, NP0030901, NP003786 and NP003785, respectively) and SNX16 (GenBank accession no. AF305780) was performed using the ClustalW program, and the aligned sequences are shown. (C) SNX16 and its mutant forms are depicted schematically with black boxes representing the PX domain. Amino acid positions within each construct are indicated by the numbers. (D) COS-7 cells were transiently transfected with FLAG-tagged SNX16 and its mutants followed 24-30 hours later by preparation of total cell lysates (Total), cytosolic fractions (cytosol), and membrane particulates (membrane), as described in the Materials and Methods. The distribution of FLAG-SNX16 and its mutants were determined in each fraction by immunoblotting with anti-FLAG antibody. (E) GFP-tagged SNX16 and its point mutant form in PX domain (green) were expressed in COS-7 cells. Cells were stained for endogenous EEA1 and transferrin receptor (TfR) with anti-EEA1 and anti-TfR primary antibody and visualized with Rhodamine Red-conjugated anti-rabbit IgG secondary antibody (red).