Self-assembly and binding of a sorting nexin to sorting endosomes

The fate of endocytosed membrane proteins and luminal contents is determine d by a materials processing system in sorting endosomes, Endosomal retentio n is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling, We report that a sorting nexin SNX1, a ca ndidate endosomal retention protein, self-assembles in vitro and in vivo, a nd has this property in common with its yeast homologue Vps5p, A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by si ze-exclusion chromatography indicates that in cytosol SNX1 tetramers are pa rt of a larger complex with additional proteins. An endosomal retention fun ction would require that SNX1 bind to endosomal membranes, yet the complexe s that we analyzed were largely soluble and little SNX1 was found in pellet fractions. Using green fluorescent protein fusions, endocytic compartment markers and fluorescence recovery after photobleaching, we found that there is an equilibrium between free cytoplasmic and early/sorting endosome-boun d pools of green fluorescent protein-SNX1. Fluorescence resonance energy tr ansfer indicated that spectral variants of green fluorescent protein-SNX1 w ere oligomeric in vivo. In cell extracts, these green fluorescent protein-S NX1 oligomers corresponded to tetrameric and larger complexes of green fluo rescent protein-SNX1, Using video microscopy, we observed small vesicle doc king and tubule budding from large green fluorescent protein-SNX1 coated en dosomes, which are features consistent with their role as sorting endosomes .