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).
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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.
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Supplementary Figure 1 Phylogenetic analysis of Syndetin orthologs.
An initial BLASTP search revealed that the first 492 amino acids of human Syndetin comprised the largest conserved domain (DUF 2450). This sequence was used in two consecutive rounds of PSI-BLAST on genomes from different organisms. Orthologs were defined as (i) being large proteins (>500 amino acids in all super-groups, with the exception of some smaller proteins in the SAR/CCTH super-group), (ii) having a high α-helical content, (iii) having either DUF 2450 or DUF 2451 domains, and (iv) yielding human Syndetin and not Vps54 as one of three top hits in reverse PSI-BLAST searches. Listed are representative species, NCBI reference sequences and length of the Syndetin orthologs.
Supplementary Figure 2 Vps54 and Syndetin exhibit distinct organelle targeting and retrograde transport activities.
(a) Co-localization of Vps54-EGFP and Syndetin-EGFP with Ang2–13Myc in rat hippocampal neurons analyzed with antibodies to GFP, the myc epitope and MAP2 as described in the legend to Figure 3. Bars, 10 μm. (b) Syndetin KD does not affect retrograde transport from endosomes to the TGN. Control (mock), Ang2-, Vps54- and Syndetin-KD HeLa cells were treated with 0.5 μg/ml Cy3-conjugated B-subunit of Shiga toxin (STxB) at 37 °C for 15 min and chased for 1 h before fixation and immunostaining. Nocodazole treatment was applied at 10 μM for 2 h following the chase where indicated. Giantin (medial Golgi marker) and TGN46 (TGN marker) were visualized by immunostaining with rabbit antibody to Giantin and sheep antibody to human TGN46 followed by Alexa647 donkey anti-rabbit and Alexa488 donkey anti-sheep antibodies. Bars, 10 μm.
Supplementary Figure 3 Syndetin KD does not affect initial Tf uptake but increases Tf accumulation over time.
(a,b) HeLa cells were treated with the indicated siRNAs and incubated with Tf-Alexa568 at 37 °C for 3 min. After three washes with ice-cold PBS, cells were fixed. Images were acquired by confocal microscopy and quantified using ImageJ. Values are the mean ± SEM (n = 3 independent experiments). The numbers of cells quantified were: mock, 171; Vps54 KD, 167; Syndetin KD, 142. n.s., P = 0.86 (Mock vs. Vps54 KD) and P = 0.69 (Mock vs. Syndetin KD). (c) HeLa cells treated with the indicated siRNAs were incubated with Tf-Alexa647 at 37 °C for different times and then washed twice with ice-cold citrate buffer (pH 4.6) and three times with PBS to remove surface-bound Tf-Alexa647. Intracellular Tf-Alexa647 was quantified by FACS after detaching cells from the plates using 2 mM EDTA. At least 1x105 cells were analyzed for each group in each independent experiment. Values are the mean ± SEM (n = 3 independent experiments).
Supplementary Figure 4 Effects of Syndetin KD on the distribution of internalized Tf-biotin in subcellular fractions and on LC3-II levels.
(a) Equal numbers of HeLa cells treated with control (mock) or Syndetin siRNAs were allowed to internalize Tf-biotin for 30 min. After stripping surface-bound Tf-biotin with citrate buffer (pH 4.6), cells were chased for 30 min. Cells were lysed and fractionated by ultracentrifugation on 5–20% iodixanol gradients. Fractions were collected and analyzed by SDS-PAGE gels and immunoblotting for the indicated proteins. Tf-biotin was detected with Streptavidin-HRP polymer according to the manufacturer’s instructions. Molecular mass markers (in kDa) are indicated at left. (b) HeLa cells were treated with siRNAs targeting the proteins indicated on top and analyzed by SDS-PAGE and blotting for the proteins indicated at right. Bafilomycin A1 was applied at 250 nM for 4 h. Molecular mass markers (in kDa) are indicated at left. Uncropped images of the blots are shown in Supplementary Fig. 5c.
Supplementary Figure 5 Uncropped images of key panels in main figures.
Red boxes indicate the cropped portion of each immunoblot presented in the corresponding main figures. Ladder of molecular markers is shown on the left of each panel.
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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. (MOV 35467 kb)
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. (MOV 37196 kb)
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. (MOV 35828 kb)
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. (MOV 36558 kb)
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. (MOV 1136 kb)
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Schindler, C., Chen, Y., Pu, J. et al. EARP is a multisubunit tethering complex involved in endocytic recycling. Nat Cell Biol 17, 639–650 (2015). https://doi.org/10.1038/ncb3129
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DOI: https://doi.org/10.1038/ncb3129
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