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. 2004 Apr;165(1):111-22.
doi: 10.1083/jcb.200312034.

Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

Matthew N J Seaman  1
Affiliations

Cargo-selective endosomal sorting for retrieval to the Golgi requires retromer

Matthew N J Seaman. J Cell Biol. 2004 Apr.

Abstract

fEndosome-to-Golgi retrieval of the mannose 6-phosphate receptor (MPR) is required for lysosome biogenesis. Currently, this pathway is poorly understood. Analyses in yeast identified a complex of proteins called "retromer" that is essential for endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p. Retromer comprises five distinct proteins: Vps35p, 29p, 26p, 17p, and 5p, which are conserved in mammals. Here, we show that retromer is required for the efficient retrieval of the cation-independent MPR (CI-MPR). Cells lacking mammalian VPS26 fail to retrieve the CI-MPR, resulting in either rapid degradation of or mislocalization to the plasma membrane. We have localized mVPS26 to multivesicular body endosomes by electron microscopy, and through the use of CD8 reporter protein constructs have examined the effect of loss of mVPS26 upon the trafficking of membrane proteins that cycle between the endosome and the Golgi. The data presented here support the hypothesis that retromer performs a selective function in endosome-to-Golgi transport, mediating retrieval of the CI-MPR, but not furin.

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Figures

Figure 1.
Figure 1.
Immunofluorescence localization of mVPS26. (A) HeLaM cells stably transfected with the respective CD8 reporter were fixed and labeled with polyclonal anti-mVPS26 and monoclonal anti-CD8 followed by secondary antibodies. Bar, 20 μm. (B) HeLaM cells stably expressing GFP-rab proteins were fixed and labeled with antisera against mVPS26 followed by fluorescently labeled secondary antibodies. Bar, 20 μm. Arrows indicate coincident labeling.
Figure 2.
Figure 2.
Electron microscopic localization of mVPS26 and Snx1. HeLaM cells grown in 6-cm dishes were permeabilized by rapid freeze/thaw. After fixation, mVPS26 or Snx1 was labeled with polyclonal antisera followed by 10-nm colloidal gold anti–rabbit. The cells were embedded in Epon and then sectioned to produce ∼50-nm-thick sections. mVPS26 (a–c) and Snx1 (d and e) are found on multivesicular bodies characteristic of endosomal compartments. Bar, 250 nm. Arrows indicate Snx1 localization to tubular–vesicular structures.
Figure 3.
Figure 3.
Loss of mVPS26 expression results in increased cell surface CI-MPR. (A) Cells grown in 6-cm dishes were labeled with [35S]methionine continuously for 3 h before lysis. mVPS26 or actin was immunoprecipitated and then subjected to SDS-PAGE and fluorography. Lane 1, wild-type cells; lane 2, mVPS26−/− cells; lane 3, control cells; lane 4, mVPS26 knock-down cells. (B) Cells were fixed and labeled with antibodies against the CI-MPR. Arrows indicate CI-MPR localization to the plasma membrane. Bars, 20 μm. (C) Cells grown in 9-cm dishes were labeled overnight with [35S]methionine. After removal from the dish, the cells were treated with proteinase K and then were precipitated and lysed. The CI-MPR, TfnR, and Snx1 were recovered from the resulting lysates and subjected to SDS-PAGE and fluorography. (D) Control or mVPS26 knock-down cells were fixed and labeled with antibodies against EEA1, mVPS26, or the CI-MPR. Arrows indicate coincident labeling. Bar, 20 μm.
Figure 4.
Figure 4.
Steady-state levels of the CI-MPR are affected after loss of mVPS26. (A) Lysates from the wild-type and mVPS26−/− mouse cells and lysates from the control and mVPS26 knock-down HeLaM cells were prepared and electrophoresed on a 10% polyacrylamide gel. The gel was stained with Coomassie blue. (B) Similar gels to the one shown in A were transferred onto nitrocellulose and then were Western blotted with various antibodies followed by 125I-protein A. Lane 1, wild-type cells; lane 2, mVPS26−/− cells; lane 3, control cells; lane 4, mVPS26 knock-down cells.
Figure 5.
Figure 5.
The CI-MPR is unstable in the mVPS26 −/− cells. (A) Cells grown in 3-cm dishes were pulse labeled with [35S]methionine for 1 h and then chased for 0, 1, 2, or 3 h. The cells were lysed and the CI-MPR was recovered by immunoprecipitation and then subjected to SDS-PAGE and fluorography. The signal on the resulting film was quantified and expressed as a percentage of the signal at time 0. The data from four experiments were averaged together and are shown in the graph. The error bars are SDs. The bottom panels are representative of the data obtained and show the instability of the CI-MPR in the mVPS26−/− cells. (B) The CI-MPR stability experiment was repeated as described above using control and mVPS26 knock-down cells. There is no apparent instability of the CI-MPR after mVPS26 knock down.
Figure 6.
Figure 6.
Loss of mVPS26 results in a defect in cathepsin D maturation. Cells grown in 3-cm dishes were pulse labeled with [35S]methionine for 15 min and then chased for 0, 1, 2, or 3 h. At the end of the chase, the media was removed and the cells were lysed. Cathepsin D was immunoprecipitated from both the intracellular (I) and extracellular (E) (media) fractions and subjected to SDS-PAGE and fluorography.
Figure 7.
Figure 7.
CD8 reporter localization and Golgi fragmentation after loss of mVPS26 expression. (A) Cells that had been treated with mVPS26 knock-down siRNA were fixed and labeled with antibodies against CD8 and mVPS26 (as in Fig. 1 A). Bar, 20 μm. (B) Control cells and mVPS26 knock-down cells labeled with anti-GM130 and anti-TGN46. Bar, 20 μm.
Figure 8.
Figure 8.
Antibody uptake experiments demonstrate a defect in the kinetics of endosome-to-TGN retrieval after mVPS26 knock down. (A) Control cells expressing the CD8–CI-MPR were incubated with anti-CD8 antibodies, washed, and then warmed to 37°C for 8, 16, or 24 min before fixation. The cells were then labeled with antibodies against mVPS26 followed by secondary antibodies. (B) mVPS26 knock-down cells were treated as in A. (C) Cells from A and B that had been chased for 24 min were labeled with antibodies against TGN46. (D) Cells from A and B that had been chased for 24 min were labeled with antibodies against Snx1. (E) CD8-sortilin–expressing cells were subjected to the anti-CD8 uptake assay. After 24 min of chase, the cells were fixed and labeled with antibodies against mVPS26 followed by secondary antibodies. Arrows in A and D indicate coincident labeling. Bars, 20 μm.
Figure 8.
Figure 8.
Antibody uptake experiments demonstrate a defect in the kinetics of endosome-to-TGN retrieval after mVPS26 knock down. (A) Control cells expressing the CD8–CI-MPR were incubated with anti-CD8 antibodies, washed, and then warmed to 37°C for 8, 16, or 24 min before fixation. The cells were then labeled with antibodies against mVPS26 followed by secondary antibodies. (B) mVPS26 knock-down cells were treated as in A. (C) Cells from A and B that had been chased for 24 min were labeled with antibodies against TGN46. (D) Cells from A and B that had been chased for 24 min were labeled with antibodies against Snx1. (E) CD8-sortilin–expressing cells were subjected to the anti-CD8 uptake assay. After 24 min of chase, the cells were fixed and labeled with antibodies against mVPS26 followed by secondary antibodies. Arrows in A and D indicate coincident labeling. Bars, 20 μm.
Figure 9.
Figure 9.
mVPS26 knock down results in a selective endosome-to-Golgi retrieval defect. (A) Control cells expressing the CD8 reporter chimeras were incubated at 37°C continuously for 3 h with the anti-CD8 antibody. After washes, the cells were fixed and then labeled as in Fig. 8, A and B. (B) As in A, but with mVPS26 knock-down cells. (C) Cells expressing both GFP-furin and CD8–CI-MPR were used in an antibody uptake experiment similar to those in Fig. 8. After 24 min of chase, the cells were fixed and labeled with secondary antibodies. Bars, 20 μm.
Figure 9.
Figure 9.
mVPS26 knock down results in a selective endosome-to-Golgi retrieval defect. (A) Control cells expressing the CD8 reporter chimeras were incubated at 37°C continuously for 3 h with the anti-CD8 antibody. After washes, the cells were fixed and then labeled as in Fig. 8, A and B. (B) As in A, but with mVPS26 knock-down cells. (C) Cells expressing both GFP-furin and CD8–CI-MPR were used in an antibody uptake experiment similar to those in Fig. 8. After 24 min of chase, the cells were fixed and labeled with secondary antibodies. Bars, 20 μm.
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