Self-assembly and binding of a sorting nexin to sorting endosomes.
The fate of endocytosed membrane proteins and luminal contents is determined by a materials processing system in sorting endosomes. Endosomal retention is a mechanism that traps specific proteins within this compartment, and thereby prevents their recycling. We report that a sorting nexin SNX1, a candidate endosomal retention protein, self-assembles in vitro and in vivo, and has this property in common with its yeast homologue Vps5p. A comparison of SNX1 expressed in bacterial and in mammalian systems and analyzed by size-exclusion chromatography indicates that in cytosol SNX1 tetramers are part of a larger complex with additional proteins. An endosomal retention function would require that SNX1 bind to endosomal membranes, yet the complexes 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-bound pools of green fluorescent protein-SNX1. Fluorescence resonance energy transfer indicated that spectral variants of green fluorescent protein-SNX1 were oligomeric in vivo. In cell extracts, these green fluorescent protein-SNX1 oligomers corresponded to tetrameric and larger complexes of green fluorescent protein-SNX1. Using video microscopy, we observed small vesicle docking and tubule budding from large green fluorescent protein-SNX1 coated endosomes, which are features consistent with their role as sorting endosomes. http://www.biologists.com/JCS/movies/jcs2058.html
Kurten
R C
RC
Department of Physiology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205-0750, USA. KurtenRichardC@exchange.uams.edu
Eddington
A D
AD
Chowdhury
P
P
Smith
R D
RD
Davidson
A D
AD
Shank
B B
BB
D000595:Amino Acid Sequence; D000818:Animals; D001704:Biopolymers; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins
D001704:Biopolymers; D002352:Carrier Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins