A bipartite sorting signal ensures specificity of retromer complex in membrane protein recycling

Abstract

Retromer is an evolutionarily conserved protein complex, which sorts functionally diverse membrane proteins into recycling tubules/vesicles from the endosome. Many of the identified cargos possess a recycling signal sequence defined as ØX[L/M/V], where Ø is F/Y/W. However, this sequence is present in almost all proteins encoded in the genome. Also, several identified recycling sequences do not follow this rule. How then does retromer precisely select its cargos? Here, we reveal that an additional motif is also required for cargo retrieval. The two distinct motifs form a bipartite recycling signal recognized by the retromer subunits, Vps26 and Vps35. Strikingly, Vps26 utilizes different binding sites depending on the cargo, allowing retromer to recycle different membrane proteins. Thus, retromer interacts with cargos in a more complex manner than previously thought, which facilitates precise cargo recognition.

Figure 1.

The FGEIRL motif of Vps10 is also important for its recycling. (A) A model of Vps10 recycling. (B) Schematic of Vps10. (C, E, G, and I) Vps10-GFP localization. (D, F, and H) Schematic of Vps10 mutational analysis and the percentage of each category of Vps10-GFP mutant localization from E (D), from G and Fig. S1 B (F), or from I and Fig. S1 C (H). Because the 1426th residue of Vps10 is Ala, A1426 was not substituted. ND, not determined; vac., vacuole. For all quantification shown in this figure, at least 30 cells were classified and the data were obtained from three independent experiments. Error bars represent SD. Scale bars: 2 µm.

Figure 2.

Two distinct sequences of Vps10 serve as a bipartite recycling signal. (A) Schematic of Vps10. (B) Weblogo residue conservation of 1428-FGEIRL-1433 and 1492-YSSL-1495 of Vps10 among 85 Vps10 homologues from related species. (C) Vps10-GFP localization. (D) The percentage of each category of Vps10-GFP mutant localization from C. vac., vacuole. (E) The Vps26-Vps10 interaction in Vps10-GFP mutants. Vps26-FLAG was immunoprecipitated (IP) from cells expressing Vps10-GFP or Vps10-GFP with a mutation in a bipartite recycling signal (Vps10^10xAla-GFP), and interacting Vps10-GFP was detected by immunoblotting using antibodies against FLAG, GFP, and G6PDH. (F) CPY sorting in vps10Δ cells expressing Vps10-GFP mutants. CPY sorting is examined by immunoblotting against CPY and Pgk1. Mature form of CPY in WT was set to 100%. Ex. and Int., extracellular and intracellular space, respectively; p2CPY, p2 precursor form of CPY; mCPY, mature CPY. (G) Ear1^ΔC-GFP and Ear1^ΔC-Vps10^C-tail-GFP localization. For all quantification shown in this figure, at least 30 cells were classified and the data were obtained from three independent experiments. Error bars represent SD. Scale bars: 2 µm.

Figure 3.

Two separate sequences are also important for Ear1 retrieval. (A, C, and E) Schematic of Ear1-mNeonGreen mutational analysis and the percentage of each category of Ear1-mNeonGreen mutant localization in rsp5(G747E) mutants from B and Fig. S1 G (A), from D and Fig. S1 H (C), or from F, G, and Fig. S1, I and J (E). vac., vacuole. (B, D, F, and G) Ear1-mNeonGreen localization in rsp5 (G747E) mutants. FL, full-length. (H) Ear1-pHluorin localization in cells expressing WT Rsp5. (J) Vps10^ΔC-GFP and Vps10^ΔC-Ear1^C-tail-GFP localization. (I) Weblogo residue conservation of 453-PPGFEF-458 and 473-INL-475 of Ear1 among 37 Ear1 homologues from related species. (K) Schematic of Ear1. For all quantification shown in this figure, at least 30 cells were classified and the data were obtained from three independent experiments. Error bars represent SD. Scale bars: 2 µm.

Figure 4.

The retromer complex recognizes the endosomal cargo through Vps26 and Vps35. (A) Model of the retromer complex in yeast. (B) The retromer assembly in retromer subunit mutants. Vps5-FLAG was immunoprecipitated (IP) from cells lacking Vps17, Vps26, Vps29, or Vps35, and interacting Vps17-HA, Vps26, Vps29, and Vps35 were detected by immunoblotting using antibody against FLAG, HA, Vps26, Vps29, Vps35, and Pgk1. (C) The localization of Vps10-GFP or Ear1-pHluorin (Ear1-pH) in WT, vps26Δ, vps29Δ, and vps35Δ cells. (D) The localization of Vps10-GFP or Ear1-pHluorin (Ear1-pH) in WT, vps5Δ, vps17Δ, and vps35Δ cells under osmotic stress. Cells expressing Vps10-GFP grown in YPD media were resuspended in water and incubated for 30 min. (E) The Vps26-Vps10 interaction in retromer subunit mutants. Vps26-FLAG was immunoprecipitated from Vps10-GFP–expressing cells lacking Vps5, Vps17, Vps29, or Vps35, and interacting Vps10-GFP was detected by immunoblotting using antibody against FLAG, GFP, and G6PDH. (F) The Vps35-Vps10 interaction in vps26Δ cells. Vps35-FLAG was immunoprecipitated from WT or vps26Δ cells, and interacting Vps10-GFP was detected by immunoblotting using antibody against FLAG, GFP, and G6PDH. Scale bars: 2 µm.

Figure 5.

Different sites in Vps26 are required for Vps10 or Ear1 recognition. (A) The localization of Vps10-GFP in vps26 mutants. (B) The percentage of each category of Vps10-GFP mutant localization from A. vac, vacuole. (C) The localization of Ear1-mNeonGreen (Ear1-mNG) in rsp5(G747E) vps26 mutants. (D) The percentage of each category of Ear1-mNeonGreen mutant localization from C. (E) The retromer assembly in vps26 mutants. Vps5-FLAG was immunoprecipitated (IP) from cells expressing vps26 mutants, and interacting Vps17-HA, Vps26, Vps29, and Vps35 were detected by immunoblotting using antibody against FLAG, HA, Vps26, Vps29, Vps35, and Pgk1. (F) The Vps5-Vps10 interaction in vps26 mutants. Vps5-FLAG was immunoprecipitated from cells expressing vps26 mutants, and interacting Vps10-GFP was detected by immunoblotting using antibody against FLAG, GFP, and G6PDH. (G) The model of retromer cargo recognition. For all quantification shown in this figure, at least 30 cells were classified and the data were obtained from three independent experiments. Error bars represent SD. Scale bars: 2 µm.