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A PX-BAR protein Mvp1/SNX8 and a dynamin-like GTPase Vps1 drive endosomal recycling

  1. Sho W Suzuki
  2. Akihiko Oishi
  3. Nadia Nikulin
  4. Jeff R Jorgensen
  5. Matthew G Baile
  6. Scott D Emr  Is a corresponding author
  1. Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, United States
Research Article
Cite this article as: eLife 2021;10:e69883 doi: 10.7554/eLife.69883
12 figures, 1 table and 2 additional files

Figures

Figure 1 with 1 supplement
The endosomal localization of Vps55 requires Mvp1.

(A) Schematic of SNX-BAR proteins in yeast. (B) Model of endosomal recycling pathways in yeast. (C) Schematic of Vps55 and OB-RGRP. (D) Vps55-GFP localization. The mCherry-Pep12 serves as an endosomal marker. (E) Vps55-GFP localization in wild-type (WT), vps35Δ (retromer mutant), snx4Δ (Snx4 complex mutant), and mvp1Δ. (F) Quantification of Vps55-GFP localization from three independent experiments. N = >30 cells. Scale bar: 1 µm.

Figure 1—source data 1

Source data associated with Figure 1F.

https://cdn.elifesciences.org/articles/69883/elife-69883-fig1-data1-v2.xlsx
Figure 1—source data 2

The list of localization altered in retromer mutants. The list of endosomal transmembrane proteins whose localization was examined in vps35Δ cells.

https://cdn.elifesciences.org/articles/69883/elife-69883-fig1-data2-v2.docx
Figure 1—figure supplement 1
The localization of endosomal membrane proteins.

(A) Schematic of retromer and Snx4 complexes. (B) The localization of mNeonGreen-Pep12 in wild-type (WT) and vps35Δ cells. (C) Vps55-GFP localization with Sec7-mCherry serving as a marker for the trans-Golgi. (D) Manders’ coefficients of Vps55-GFP with Sec7-mCherry or mCherry-Pep12 from C and Figure 1D. (E) Vps55-GFP localization in snx41Δ, snx42Δ, and ykr078wΔ cells. Scale bar: 1 µm.

Figure 2 with 2 supplements
Mvp1 is an endosomal coat complex for Vps55 recycling.

(A) Mvp1-GFP localization. The mCherry-Pep12 serves as an endosomal marker. (B) Schematic of Mvp1 mutants. (C) The localization of Mvp1-GFP mutants. (D) The localization of Vps55-GFP mutants. (E) Quantitation of Vps55-GFP localization of mvp1 mutants from three independent experiments. N = >30 cells. (F) The binding of SNX-BAR proteins with Mvp1. FLAG-tagged SNX-BAR proteins were immunoprecipitated (IP) from cells expressing Mvp1-GFP, and the IP products were analyzed by immunoblotting using antibodies against FLAG, green fluorescent protein (GFP), and glucose-6-phosphate dehydrogenase (G6PDH). (G) The dimer formation of mvp1 mutants. Mvp1-FLAG was immunoprecipitated from cells expressing Mvp1-GFP mutants, and the IP products were analyzed by immunoblotting using antibodies against FLAG, GFP, and G6PDH. (H) Live-cell imaging analysis of Vps55-mNeonGreen and mCherry-Vps21. (I) Live-cell imaging analysis of Mvp1-mNeonGreen and mCherry-Vps21. Scale bar: 1 µm.

Figure 2—figure supplement 1
The analysis of Mvp1.

(A) Mvp1-GFP localization in pep12Δ cells. Scale bar: 1 µm. (B) Mutation sites used in this study are shown on the cryo-electron microscopy (cryo-EM) structure of Saccharomyces cerevisiae Mvp1 (PDB ID code: 6Q0X). (C) Sequence comparison of the residue required for phosphatidylinositol 3-phosphate (PI3P) binding among Phox homology (PX) domain-containing proteins. (D) Line scan analysis performed for the region highlighted by the white line from Figure 2H.

Figure 2—video 1
Vps55-mNeonGreen decorated tubule emerged and detached from the endosome.

Cells expressing Vps55-mNeonGreen (green) and mCherry-Vps21 (red; endosome). Cells were imaged every 1 s.

Figure 3 with 1 supplement
Mvp1 recognizes Vps55 through a specific sorting motif.

(A, B, and D) Schematic of Vps55 mutational analysis and quantitation of Vps55-GFP mutant localization, from Figure 3—figure supplement 1A (A), Figure 3—figure supplement 1B (B), and Figure 3—figure supplement 1C (D). (C) The localization of Vps55-GFP mutants. (E) The Mvp1-Vps55 interaction in Vps55-FLAG mutants. Vps55-FLAG mutants were immunoprecipitated (IP) from cells expressing Mvp1-GFP, and the IP products were analyzed by immunoblotting using antibodies against FLAG, green fluorescent protein (GFP), and glucose-6-phosphate dehydrogenase (G6PDH). (F) Schematic of Vps55 and the residues facilitating its interaction with Mvp1. (G) The localization of overexpressed Vps55-GFP. (H) Vps55-GFP sorting in vacuolar hydrolases (pep4Δ and pep4Δ prb1Δ) and ESCRT (vps4Δ) mutants. Cell lysates expressing Vps55-GFP were analyzed by immunoblotting using antibodies against GFP and G6PDH. (I) The ubiquitination of overexpressed Vps55-GFP. Overexpressed Vps55-GFP was immunoprecipitated from yeast cells under denaturing conditions, and the IP products were analyzed by immunoblotting using antibodies against GFP and ubiquitin. (J) Vps55-GFP-Ub localization in ESCRT mutants. (K) Model of Vps55 recycling and degradation at the endosome. For all quantification shown in this figure, n = >30 cells from three independent experiments. Scale bar: 1 µm.

Figure 3—figure supplement 1
The analysis of Vps55.

(A and B) Localization of Vps55-GFP mutants. In K60A and D65A mutants, the excitation laser intensity was lowered to 20%, because its expression was higher than other mutants. (C) The Mvp1-Vps55 interaction in vam3Δ cells. Vps55-FLAG mutants were immunoprecipitated (IP) from vam3Δ cells expressing Mvp1-GFP, and the IP products were analyzed by immunoblotting using antibodies against FLAG, green fluorescent protein (GFP), and glucose-6-phosphate dehydrogenase (G6PDH). (D) Immunoblotting of cells expressing increasing amounts of Vps55-GFP. (E) The localization of overexpressed Vps55-GFP and Vps68. (F) Quantification of Vps55-GFP localization from three independent experiments. N = >30 cells. (G) Cell lysates expressing Vps55-GFP or Vps55-GFP-Ub were analyzed by immunoblotting using antibodies against GFP and G6PDH. Scale bar: 1 µm.

Figure 4 with 2 supplements
Mvp1 recruits dynamin-like GTPase Vps1 to catalyze membrane scission.

(A) Schematic of Vps1. (B) Vps55-mNeonGreen localization in WT and vps1Δ cells. (C) Quantitation of Vps55-mNeonGreen localization, from B. (D) The live-cell imaging of Vps1-GFP. The mCherry-Vps21 serves as an endosomal marker. (E) Model of Dynamin-1-mediated membrane fission. (F) Sequence comparison of residues required for guanosine-5'-triphosphate (GTP) hydrolysis in Homo sapiens Dynamin-1, H. sapiens Dynamin-2, H. sapiens Dynamin-3, Saccharomyces cerevisiae Dnm1, and S. cerevisiae Vps1. (G) Vps55-mNeonGreen localization in vps1K42A mutants. (H) Quantitation of Vps55-mNeonGreen localization, from G. (I) The localization of Mvp1-mNeonGreen, mCherry-Vps21, and Vps1K42A-BFP. (J) Vps1-GFP localization in wild-type (WT) and vps35Δ snx4Δ mvp1Δ triple mutants. (K) Quantitation of Vps1-GFP localization, from J and Figure 4—figure supplement 1F. (L) The Mvp1-Vps1 interaction. Mvp1-FLAG was immunoprecipitated (IP) from cells expressing Vps1-GFP mutants, and the IP products were analyzed using antibodies against FLAG, green fluorescent protein (GFP), and glucose-6-phosphate dehydrogenase (G6PDH). (M) In vitro binding assay between Mvp1 and Vps1-GFP. The proteins bound to anti-GFP magnetic beads were detected by Coomassie staining. For all quantifications shown in this figure, n = >30 cells from three independent experiments. Scale bar: 1 µm.

Figure 4—figure supplement 1
The analysis of Vps1.

(A) Growth of Vps1-GFP-expressing cells. (B, C, and D) Vps1-GFP localization with Sec7-mCherry serving as a marker for the trans-Golgi (B), Nhx1-mCherry serving as a marker for the endosome (C), or Mvp1-mRFP (D). (E) Schematic of Dynamin-1 and Vps1 domains and mutated residues. (F) Vps1-GFP localization in wild-type (WT), vps35Δ, snx4Δ, and mvp1Δ cells. (G) Vacuole morphology in vps35Δ, snx4Δ, and mvp1Δ supplemented with 2 mM choline or 2 mM ethanolamine. (H) Vps1-GFP localization in WT or vps34Δ cells. (I) Quantification of Vps1-GFP localization from H. (J) Mvp1-mNeonGreen localization in WT and vps1Δ cells with mCherry-Vps21 serving as an endosomal marker. (K) Localization of Mvp1-GFP in vps1Δ cells. (L) Quantification of Mvp1-GFP localization from K. (M) Vps1-GFP localization in WT, vps35Δ, snx4Δ, and mvp1Δ cells expressing Mvp1 mutants. (N) Sequence comparison of residues required for the assembly of Vps1 in Homo sapiens Dynamin-1, H. sapiens Dynamin-2, H. sapiens Dynamin-3, Saccharomyces cerevisiae Dnm1, and S. cerevisiae Vps1. Scale bar: 1 µm.

Figure 4—video 1
The Vps1-GFP punctate structures on the endosome were elongated and then divided.

Cells expressing Vps1-GFP (green) and mCherry-Vps21 (red; endosome). Cells were imaged every 1 s.

Figure 5 with 1 supplement
Mvp1 mainly mediates retromer-independent endosomal recycling.

(A) Localization of Vps10-GFP in wild-type (WT), vps35Δ, and mvp1Δ cells. (B) Quantification of Vps10-GFP, Kex2-GFP, and GFP-Neo1 localization from A and Figure 5—figure supplement 1E,F, respectively. N = >30 cells from three independent experiments. (C) Schematic for immunoisolation of Vps55-FLAG-containing structures. (D) The immunoisolation of Vps55-containing vesicles. Vps55-FLAG-containing structures were immunoisolated from sec18ts mutants incubated at 37°C for 1 hr, and the isolated structures were analyzed by immunblotting using antibodies against FLAG, Vps10 (retromer cargo), Vps21 (endosome), Pho8 (vacuole), and glucose-6-phosphate dehydrogenase (G6PDH) (cytoplasm). (E) Electron microscopy (EM) analysis of the isolated Vps55-FLAG-containing structures from D. (F) Live-cell imaging analysis of Vps55-mNeonGreen and Vps10-mCherry. (G) Cell growth in vps35Δ snx4Δ mvp1Δ triple mutants. Cells lacking Vps35 as well as Snx4 and Mvp1 were grown at 26°C and 37°C. (H) Model of retromer-, Snx4-, and Mvp1-mediated recycling. (I) Nhx1 localization in SNX-BAR mutants. (J) Quantitation of Nhx1-GFP localization, from I and Figure 5—figure supplement 1M. N = >30 cells from three independent experiments. Scale bar: 1 µm.

Figure 5—figure supplement 1
The analysis of the retromer pathway in Mvp1 mutants.

(A) The association of Mvp1 with the retromer complex. Vps5-FLAG was immunoprecipitated (IP) from cells expressing Vps17-HA, Vps26-Myc, and Mvp1-GFP, and the IP products were analyzed by immunoblotting using antibodies against FLAG, hemagglutinin (HA), Myc, Vps29, Vps35, green fluorescent protein (GFP), and glucose-6-phosphate dehydrogenase (G6PDH). (B) Mvp1 binding to the retromer subunits. Mvp1-FLAG was immunoprecipitated from cells expressing Vps17-HA, and the IP products were analyzed by immunoblotting using antibodies against Vps5, HA, Vps26, Vps29, Vps35, and G6PDH. (C and D) Localization of GFP-Neo1 with Sec7-mCherry as a marker for the trans-Golgi (C) and Nhx1-mCherry as a marker for the endosome (D). (E and F) Localization of Kex2-GFP (E) and GFP-Neo1 (F) in wild-type (WT), vps35Δ, and mvp1Δ cells. (G) Hypothesis of Mvp1-mediated recycling. (H) Vps55-GFP localization in sec18ts mutants at 26°C or 37°C for 60 min. (I) Electron microscopy (EM) analysis of the isolated Vps55-FLAG-containing structures. Vps55-FLAG-containing structures were immunoisolated from sec18ts mutants incubated at 37°C for 1 hr, and then eluated using FLAG peptides. Eluated products were analyzed by negative-stain EM. (J) Immunoisolation of Vps10-containing vesicles. Vps10-FLAG-containing structures were immunoisolated from sec18ts mutants incubated at 37°C for 1 hr, and the isolated structures were analyzed by immunblotting using antibodies against FLAG, GFP, and G6PDH. (K) Live-cell imaging of Vps55-mNeonGreen and Vps10-mCherry. (L) GFP-Snc1 localization in WT, snx4Δ, and mvp1Δ cells. (M) Nhx1 localization in vps35Δ snx4Δ, vps35Δ mvp1Δ, and snx4Δ mvp1Δ cells. Scale bar: 1 µm.

Figure 6 with 1 supplement
Retromer, Snx4, and Mvp1 complexes are required for proper function of the endosome.

(A) Schematic of Mup1-pHluorin sorting. (B) Mup1-pHluorin localization in wild-type (WT) and vps35Δ snx4Δ mvp1Δ cells. Cells expressing Mup1-pHluorin were grown to mid-log phase and stimulated with 20 µg/ml methionine. Scale bar: 1 µm. (C) Mup1-pHlourin processing in WT and vps35Δ snx4Δ mvp1Δ cells. Mup1 sorting was stimulated as in B. (D) GFP-CPS sorting in WT and vps35Δ snx4Δ mvp1Δ cells. (E) Thin-section electron microscopy (EM) of an endosome in vps35Δ snx4Δ mvp1Δ cells. (F) Schematic of screening for multicopy suppressors of vps35Δ snx4Δ mvp1Δ triple mutants. (G) Growth of vps35Δ snx4Δ mvp1Δ triple mutants overexpressing Neo1. (H) Schematic of duramycin assay to evaluate extracellular phosphatidylethanolamine (PE). (I) Growth of recycling mutants in the presence of duramycin.

Figure 6—figure supplement 1
The analysis of triple recycling pathway mutants.

(A) Mup1-GFP sorting in SNX-BAR mutants. Mup1 sorting was stimulated as in Figure 6B. (B) Thin-section electron microscopy (EM) of endosomes in wild-type (WT) and vps35Δ snx4Δ mvp1Δ cells. (C) Summary of identified multicopy suppressors from Figure 6F. (D) Schematic for the plasma membrane (PM) integrity assay for WT and vps35Δ snx4Δ mvp1Δ triple mutants. (E) Quantitation of propidium iodide-stained cells from D.

Figure 7 with 4 supplements
Mvp1-mediated endosomal recycling is evolutionarily conserved.

(A) Schematic of Mvp1 and Snx8. (B) Immunofluorescence of GFP-SNX8-expressing Hela cells, with EEA1 serving as an endosomal marker. (C) Live-cell imaging of GFP-SNX8. (D) Live-cell imaging of highly expressed GFP-SNX8. (E) Model of Mvp1-mediated endosomal recycling. Scale bar: 1 µm.

Figure 7—figure supplement 1
The analysis of GFP-SNX8 tubule structure.

(A) Live-cell imaging of GFP-SNX8. (B) GFP-SNX8 localization of Hela cells expressing GFP-SNX8 at low or high levels. (C and D) Quantification of SNX8 puncta size (C) or max length of SNX8 tubule (D) from B. (E) GFP-SNX8 tubule structures in GFP-SNX8 highly expressing cells. Scale bar: 1 µm.

Figure 7—figure supplement 2
Highly expressed Vps55 is mislocalized in the retromer mutants.

(A) Vps55-GFP localization in cells expressing both Vps55 and Vps55-GFP. (B) Mvp1-GFP localization in wild-type (WT) and vps35Δ cells. (C) GFP-2xFYVE localization in WT and vps35Δ cells. Scale bar: 1 µm.

Figure 7—video 1
GFP-SNX8 tubule structure budded from the endosome.

Hela cells expressing GFP-SNX8. Cells were imaged every 5 s.

Figure 7—video 2
GFP-SNX8 tubules emerged from their concentration site, related to Figure 7D.

Hela cells expressing GFP-SNX8. Cells were imaged every 5 s.

Author response image 1
Author response image 2
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Author response image 4
Author response image 5

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
AntibodyAnti-DYKDDDDK (mouse monoclonal)WAKO1E6WB (1:2000)
AntibodyAnti-GFP (mouse monoclonal)RocheClone 7.1/13.1WB (1:5000)
AntibodyAnti-GFP (mouse monoclonal)Santa CruzB-2WB (1:5000)
AntibodyAnti-GFP (rabbit polyclonal)Torrey Pines BiolabsWB (1:5000)
AntibodyAnti-HA(mouse monoclonal)Roche12CA5WB (1:5000)
AntibodyAnti-Myc(mouse monoclonal)Santa Cruz9E10WB (1:5000)
AntibodyAnti-Vps5(rabbit polyclonal)Horazdovsky et al., 1997WB (1:5000)
AntibodyAnti-Vps26(rabbit polyclonal)Reddy and Seaman, 2001WB (1:5000)
AntibodyAnti-Vps29(rabbit polyclonal)Seaman et al., 1998WB (1:5000)
AntibodyAnti-Vps35(rabbit polyclonal)Seaman et al., 1998WB (1:5000)
AntibodyAnti-Vps10(mouse monoclonal)AbcamWB (1:5000)
AntibodyAnti-Vps21(rabbit polyclonal)Horazdovsky et al., 1994WB (1:5000)
AntibodyAnti-G6PDH(rabbit polyclonal)Sigma-AldrichWB (1:20,000)
AntibodyAnti-ALP(mouse monoclonal)NOVEXWB (1:1000)
AntibodyAnti-Pgk1(mouse monoclonal)InvitrogenWB (1:10,000)
AntibodyAnti-EEA1(rabbit monoclonal)Cell Signaling TechnologyC45B10IF (1:300)
AntibodyAnti-Ubiquitin(mouse monoclonal)Santa Cruz BiotechnologyP4D1WB (1:500)
AntibodyIRDye 800CW goat anti-mouseLI-CORWB (1:5000)
AntibodyIRDye 800CW goat anti-rabbitLI-CORWB (1:5000)
AntibodyIRDye 680LT goat anti-rabbitLI-CORWB (1:5000)
AntibodyIRDye 680LT goat anti-mouseLI-CORWB (1:5000)
AntibodyGoat Alexa Fluor Plus 647 anti-rabbitThermo Fisher ScientificIF (1:250)
OtherN-ethylmaleimideAcros Organics156100050
OtherCompleteProtease Inhibitor CocktailRoche11697498001
OtherNHS beadsTAMAGAWA SEIKITAS8848 N1141
Other3 X FLAG PeptideSigmaF4799-25MG
OtherConcanavalin ASigmaL7647-250MG
OtherGFP-TRAP_A beadsChromo Tekgta-10
OtherFuGENE HD Transfection ReagentPromegaE2311
OtherProLong Gold Antifade MountantThermo Fisher ScientificP10144
OtherPMSFSigma10837091001
OtherSaponinCALBIOCHEM558,255
OtherTriton X-100SIGMAX100-500ML
OtherTALON Metal Affinity ResinClontech635,502
OtherSUMO ProteaseMiliporeSAE0067-2500UN
Software, algorithmSoftWoRxGE Healthcare
Software, algorithmSlideBook 6.0Intelligent Imaging Innovations
Software, algorithmImageJNIH
Software, algorithmSnapGeneGSL Biotech

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