Sorting in early endosomes reveals connections to docking- and fusion-associated factors

The early endosomes constitute a major sorting platform in eukaryotic cells. They receive material through fusion with endocytotic vesicles or with trafficking vesicles from the Golgi complex and later sort it into budding vesicles. While endosomal fusion is well understood, sorting is less characte...

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Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2009-06, Vol.106 (24), p.9697-9702
Main Authors: Barysch, Sina V, Aggarwal, Shweta, Jahn, Reinhard, Rizzoli, Silvio O
Format: Article
Language:English
Subjects:
Animals
Antibodies
Biochemistry
Biological Sciences
Budding
Cell membranes
Cell-Free System
Cholera
Endosomes
Eukaryotes
Freight
Lipids
Low density lipoprotein
Membrane Fusion
Molecular biology
Organelles
PC12 Cells
Rats
Recycling
Transferrin - metabolism
Transferrins
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Summary:The early endosomes constitute a major sorting platform in eukaryotic cells. They receive material through fusion with endocytotic vesicles or with trafficking vesicles from the Golgi complex and later sort it into budding vesicles. While endosomal fusion is well understood, sorting is less characterized; the 2 processes are generally thought to be effected by different, unrelated machineries. We developed here a cell-free assay for sorting/budding from early endosomes, by taking advantage of their ability to segregate different cargoes (such as transferrin, cholera toxin subunit B, and low-density lipoprotein, LDL) into different carrier vesicles. Cargo separation required both carrier vesicle formation and active maturation of the endosomes. Sorting and budding were insensitive to reagents perturbing clathrin coats, coatomer protein complex-I (COPI) coats, dynamin, and actin, but were inhibited by anti-retromer subunit antibodies. In addition, the process required Rab-GTPases, phosphatidylinositol-3-phosphate, and, surprisingly, the docking factor early endosomal autoantigen 1 (EEA1). Sorting also required the function of the N-ethylmaleimide-sensitive factor (NSF), a well-known fusion cofactor, while it did not depend on preceding fusion of endosomes. We conclude that fusion, docking, and sorting/budding are interconnected at the molecular level.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0901444106