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EARP is a multisubunit tethering complex involved in endocytic recycling

Nature Cell Biology volume 17, pages 639650 (2015) | Download Citation

Abstract

Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In particular, it is unknown if endocytic recycling requires the function of multisubunit tethering complexes, as is the case for other intracellular trafficking pathways. Herein we describe a tethering complex named endosome-associated recycling protein (EARP) that is structurally related to the previously described Golgi-associated retrograde protein (GARP) complex. The two complexes share the Ang2, Vps52 and Vps53 subunits, but EARP contains an uncharacterized protein, syndetin, in place of the Vps54 subunit of GARP. This change determines differential localization of EARP to recycling endosomes and GARP to the Golgi complex. EARP interacts with the target SNARE syntaxin 6 and various cognate SNAREs. Depletion of syndetin or syntaxin 6 delays recycling of internalized transferrin to the cell surface. These findings implicate EARP in canonical membrane-fusion events in the process of endocytic recycling.

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Acknowledgements

We thank X. Zhu and N. Tsai for technical assistance, J. Presley for discussions and R. Mattera and D. Gershlick for critical review of the manuscript. This work was funded by the Intramural Program of NICHD, NIH (ZIA HD001607).

Author information

Author notes

    • Christina Schindler
    •  & Yu Chen

    These authors contributed equally to this work.

Affiliations

  1. Cell Biology and Metabolism Program, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA

    • Christina Schindler
    • , Yu Chen
    • , Jing Pu
    • , Xiaoli Guo
    •  & Juan S. Bonifacino

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Contributions

C.S., Y.C. and J.S.B. conceived the project. C.S. and Y.C. carried out most of the experiments. J.P. contributed to SNARE pulldowns and X.G. to subcellular fractionation experiments. C.S., Y.C., J.P., X.G. and J.S.B. analysed the data. J.S.B., C.S. and Y.C. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Juan S. Bonifacino.

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Videos

  1. 1.

    Differential localization of Vps54 and Syndetin in live H4 cells.

    H4 cells stably expressing Vps54-EGFP were transfected with a plasmid encoding Syndetin-mCherry and imaged by live-cell confocal microscopy after 48 h.

  2. 2.

    Co-localization of Syndetin with Rab4A observed by TIRF microscopy of live rat hippocampal neurons.

    Rat hippocampal neurons were co-transfected with plasmids encoding Syndetin-EGFP and TagRFP-Rab4A on DIV-3 and examined by TIRF microscopy on DIV-7. Inset shows magnifications of the areas boxed with solid lines.

  3. 3.

    Co-localization of Syndetin with Rab4A observed by confocal microscopy of live HeLa cells.

    Syndetin-KD HeLa cells were transiently transfected with plasmids encoding mouse Syndetin-EGFP and TagRFP-Rab4A and imaged at 37 °C after 48 h.

  4. 4.

    Low co-localization of Syndetin with Rab5A observed by confocal microscopy of live HeLa cells.

    Syndetin-KD HeLa cells were transiently transfected with plasmids encoding mouse Syndetin-EGFP and TagRFP-Rab5A and imaged at 37 °C after 48 h.

  5. 5.

    Rapid access of internalized Tf into the Syndetin compartment.

    Syndetin-KD HeLa cells were transiently transfected with plasmids encoding Syndetin-4xEGFP and, after 48 h, imaged at after addition of Tf-Alexa568 to the medium.

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https://doi.org/10.1038/ncb3129

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