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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.009654

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Identification of a novel conserved sorting motif required for retromer-mediated endosome-to-TGN retrieval

University of Cambridge, Cambridge Institute for Medical Research/Clinical Biochemistry, Wellcome Trust/MRC building, Addenbrookes Hospital, Cambridge, CB2 0XY, UK

View larger version (47K): [in this window] [in a new window]   Fig. 1. Antibody uptake is dependent upon the presence of the CD8 lumenal domain. (A) CD8-CIMPR-expressing cells were mixed with untransfected HeLa cells and incubated with anti-CD8 continuously for 3 hours. After fixation, the cells were labelled with anti-SNX1 (Snx1) antibodies and fluorescently labelled anti-mouse antibodies. The anti-CD8 antibodies are detectable only in some cells, even at high exposure. (B) CD8-CIMPR or untransfected HeLa cells (Helas) were seeded at similar density into a 24-well tissue culture dish. Four wells of each cell line were incubated with anti-CD8 for 3 hours, fixed, permeabilised and then incubated with 125I-protein-A. Four wells of each were incubated without anti-CD8 before fixation and incubation with 125I-protein-A and the other four wells were lysed in SDS-PAGE sample buffer and subjected to SDS-PAGE. The transferin receptor (TfnR) and CD8-CIMPR were detected by western blotting (shown in the inset). Only cells expressing the CD8-CIMPR and incubated with the anti-CD8 antibody bound the 125I-protein-A.

View larger version (120K): [in this window] [in a new window]   Fig. 2. Alignment of CIMPR tails and schematic diagram of the truncations of the CIMPR tail. (A) The cytoplasmic tail sequences of the CIMPR from species ranging from zebrafish through chicken, marsupials, rodents, primates and humans were aligned using the ClustalW program (http://www.ch.embnet.org/). The sequences were aligned according to homology and then the shading added using BOXSHADE (http://www.ch.embnet.org/). The numbering of the residues applies to the cow sequence which was used to make the CD8-CIMPR reporter and all of the various truncations with the exception of the 1-19LL construct, which was based upon the rat CIMPR tail sequence. The YXX and SDEDLL motifs and other regions known to bind sorting/trafficking machinery such as AP-1 are highlighted by the black bars. The alignment reveals that the most conserved region of the CIMPR tail lies in the first 100 amino acids. (B) Schematic representation of the different CD8-CIMPR truncations. The locations of the YXX and SDEDLL motifs are again indicated.

View larger version (67K): [in this window] [in a new window]   Fig. 3. Antibody uptake experiment to evaluate retrieval to the TGN. HeLa cells stably transfected with CD8-CIMPR and the various truncations were incubated with the monoclonal anti-CD8 continuously for 3 hours prior to fixation. Cells were labelled with anti-TGN46 followed by appropriate secondary antibodies. All the constructs can retrieve to the TGN with the exception of the 1-75 truncation. Bar, 20 µm.

View larger version (65K): [in this window] [in a new window]   Fig. 4. The 1-75 truncation accumulates in VPS26-positive endosomes and is unstable. (A) Cells expressing the 1-75 truncation were incubated for three hours continuously with the anti-CD8 antibody prior to fixation. Cells were labelled with anti-VPS26 antiserum followed by appropriate secondary antibodies. There is significant colocalisation of the endocytosed anti-CD8 and VPS26 (shown enlarged in the inset boxes and indicated by arrows). Bar, 20 µm. (B) Cells expressing the CD8-CIMPR and truncations were treated with 100 µg/ml cycloheximide for 0, 3 or 6 hours. Cells were then lysed and the CD8 reporter immunoprecipitated and subjected to SDS-PAGE and western blotting. The 1-75 truncation is unstable and undetectable after 3 hours of cycloheximide treatment.

View larger version (37K): [in this window] [in a new window]   Fig. 5. Alanine scanning mutagenesis of the Tail19-55 reporter. (A) Alignment of the sequence of the Tail19-55 reporter and the ten mutants derived from it. The alanines that have replaced the endogenous amino acids are shown in bold. The YXX motif is indicated and was deliberately not mutated as this sequence was likely to be necessary for the efficient endocytosis of the CD8 reporter. Only the serine at position 19 and the glycine at position 55 were left unmutated. (B) The stability of the Tail19-55 reporter and the 10 different mutants was determined by cycloheximide chase as described before. Mutant 7 was the least stable and was predominately present as the lower molecular mass form.

View larger version (106K): [in this window] [in a new window]   Fig. 6. The WLM motif is necessary for endosome-to-TGN retrieval. (A) Cells expressing the alanine scanning mutants (see Fig. 5) were incubated with anti-CD8 continuously for three hours prior to fixation and labelling with anti-TGN46 and appropriate secondary antibodies. Mutant 7 fails to retrieve to the TGN. Bar, 20 µm. (B) The mutant 7-expressing cells were treated as above but labelled with anti-VPS26 instead of anti-TGN46 followed by appropriate secondary antibodies. There is significant colocalisation between endocytosed anti-CD8 and VPS26 (shown enlarged in the inset boxes and highlighted by arrows). Bar, 20 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 7. The sortilin tail contains a sequence, FLV similar to the CIMPR WLM sequence. (A) Alignment of the sequences of the cytoplasmic tails of sortilin from various species. This alignment was performed in an identical fashion to that of the CIMPR tail (see Fig. 1). The FLV sequence is highlighted by the black bar, the sortilin YXX motif is also indicated. (B) Cells expressing the CD8-CIMPR, CD8-sortilin and the WLM-AAA and FLV-AAA mutants were analysed using the anti-CD8 uptake assay. The WLM-AAA and FLV-AAA mutants both fail to retrieve to the TGN. (C) The WLM-AAA and FLV-AAA mutants are both unstable and degraded rapidly when compared with the CD8-CIMPR and CD8-sortilin reporters respectively. Bar, 20 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 8. (A) The sortilin FLV motif can replace the CIMPR WLM motif. The WLM motif was mutated to FLV in the context of the Tail19-55 reporter. Cells expressing the Tail19-55 reporter, mutant 7 and the WLM-FLV mutant were analysed by anti-CD8 uptake assay. The WLM-FLV reporter was efficiently retrieved to the TGN to colocalise with TGN46 in a similar fashion to the Tail19-55 reporter. Bar, 20 µm. (B) The stability of the WLM-FLV reporter was determined by cycloheximide chase and found to be similar to the Tail19-55 reporter, unlike mutant 7, which was unstable and degraded.

View larger version (59K): [in this window] [in a new window]   Fig. 9. The WLM motif is necessary for the interaction with retromer. (A) Cells expressing CD8-CIMPR and various truncations were lysed and the CD8 reporter was immunoprecipitated under native conditions. The immunprecipitates were analysed by SDS-PAGE and western blotting. Retromer and AP-1 was able to co-immunoprecipitate with CD8-CIMPR, 75-163 and 56-143 but not 1-75. (B) Similar to A, cells expressing CD8 reporter were analysed by native immunoprecipitation. The CD8-WLM-AAA mutant, which has an almost full-length tail and localises to endosomes, does not co-immunoprecipitate with retromer or AP-1. (C) CD8-CIMPR cells were fixed and labelled with antibodies against VPS26 and AP-1. There is no obvious colocalisation present. Bar, 20 µm.

View larger version (64K): [in this window] [in a new window]   Fig. 10. Retromer is required for endosome-to-TGN retrieval but AP-1 is not. (A) siRNA knockdown (KD) of VPS26 or µ1A is assessed by western blotting lysates from KD cells. Loss of VPS26 results in degradation of VPS35, whereas loss of µ1A causes the 1 subunit to be degraded. The transferrin receptor (TfnR) and actin are unaffected by the siRNA knockdowns and serve as loading controls in this experiment. (B) Control or VPS26 or µ1A KD cells were analysed by antibody uptake. Loss of VPS26 results in a block in endosome-to-TGN retrieval of the CD8-CIMPR but loss of µ1A does not cause the same phenotype. (C) The CD8-CIMPR accumulates in a Snx1-positive endosome (indicated by arrows) in VPS26 KD cells, but not in µ1A KD cells. Loss of either VPS26 or µ1A resulted in cells becoming enlarged and multinucleated. Bar, 20 µm.