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

ABSTRACT: 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.

SUBMITTER: Barysch SV

PROVIDER: S-EPMC2691687 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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

Barysch Sina V SV Aggarwal Shweta S Jahn Reinhard R Rizzoli Silvio O SO

Proceedings of the National Academy of Sciences of the United States of America 20090601 24

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 th ...[more]

PMID: 19487677

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Project description:UnlabelledThe E1 membrane protein of rubella virus (RuV) is a class II membrane fusion protein structurally related to the fusion proteins of the alphaviruses, flaviviruses, and phleboviruses. Virus entry is mediated by a low pH-dependent fusion reaction through E1's insertion into the cell membrane and refolding to a stable homotrimer. Unlike the other described class II proteins, RuV E1 contains 2 fusion loops, which complex a metal ion between them by interactions with residues N88 and D136. Insertion of the E1 protein into the target membrane, fusion, and infection require calcium and are blocked by alanine substitution of N88 or D136. Here we addressed the requirements of E1 for calcium binding and the intracellular location of the calcium requirement during virus entry. Our results demonstrated that N88 and D136 are optimally configured to support RuV fusion and are strongly selected for during the virus life cycle. While E1 has some similarities with cellular proteins that bind calcium and anionic lipids, RuV binding to the membrane was independent of anionic lipids. Virus fusion occurred within early endosomes, and chelation of intracellular calcium showed that calcium within the early endosome was required for virus fusion and infection. Calcium triggered the reversible insertion of E1 into the target membrane at neutral pH, but E1 homotrimer formation and fusion required a low pH. Thus, RuV E1, unlike other known class II fusion proteins, has distinct triggers for membrane insertion and fusion protein refolding mediated, respectively, by endosomal calcium and low pH.ImportanceRubella virus causes a mild disease of childhood, but infection of pregnant women frequently results in miscarriage or severe birth defects. In spite of an effective vaccine, RuV disease remains a serious problem in many developing countries. RuV infection of host cells involves endocytic uptake and low pH-triggered membrane fusion and is unusual in its requirement for calcium binding by the membrane fusion protein. Here we addressed the mechanism of the calcium requirement and the required location of calcium during virus entry. Both calcium and low pH were essential during the virus fusion reaction, which was shown to occur in the early endosome compartment.

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

Publications

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

Similar Datasets

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