Research ArticleSTRUCTURAL BIOLOGY

Structural insights into the EGO-TC–mediated membrane tethering of the TORC1-regulatory Rag GTPases

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Science Advances  25 Sep 2019:
Vol. 5, no. 9, eaax8164
DOI: 10.1126/sciadv.aax8164
  • Fig. 1 Crystal structure of the EGOC.

    (A) Schematic diagram of the EGOC components. The interacting regions of Ego1 with Ego2, Ego3, Gtr1, and Gtr2 are indicated. The Roadblock domains and GTPase domains in Ego2, Ego3, Gtr1, and Gtr2 are also indicated. (B) Overall structure of the EGOC in two different views with Ego1 shown in pink, Ego2 in blue, Ego3 in green, Gtr1 in yellow, and Gtr2 in cyan.

  • Fig. 2 Detailed interactions among different EGOC components.

    Close-up view of Ego1 interacting with Ego2 and Ego3 (A to C), with Gtr2 (D), and with Gtr1 (E). (F) Detailed interactions of Ego3 with Gtr1-Gtr2. The residues involved in the interactions are shown in ball-and-stick models and colored as in Fig. 1B. For clarity, the residues of Ego1 are marked in black. The hydrophilic interactions are indicated with dashed lines.

  • Fig. 3 Subcellular localization and Gtr-association of Ego1 and Ego3 variants mutated on key structurally predicted interacting residues.

    (A) Localization of indicated Ego1-GFP (green fluorescent protein) alleles was examined in prototrophic ego1∆ cells grown exponentially in synthetic drop-out medium. (B) Localization of GFP-Gtr2 was assessed in prototrophic ego1gtr2∆ cells coexpressing, or not (Control), indicated Ego1-HA3 variants, and cultured as in (A). (C) Anti-GFP immunoprecipitations (IPs) were carried out on lysates from cells described in (B) or from ego1∆ cells expressing Ego1WT-HA3. Input and IP fractions were analyzed by Western blot and probed with anti-HA and anti-GFP antibodies. (D) Localization of GFP-Gtr1 was examined in prototrophic ego1gtr1∆ cells coexpressing, or not (Control), either Ego1WT-HA3 or Ego1F184A-HA3, and cultured as in (A). (E) Anti-GFP IPs were carried out on lysates from cells described in (D) or from ego1∆ cells expressing Ego1WT-HA3, and processed as in (C). (F) Localization of Ego3WT-GFP or indicated Ego3-GFP alleles was examined in prototrophic ego3∆ cells grown as in (A). (G) Localization of genomically integrated GFP-Gtr1 was assessed in prototrophic ego3gtr1∆ cells coexpressing, or not (Control), either Ego3WT-HA3 or the indicated Ego3-HA3 mutants, and cultured as in (A). (H) Anti-GFP IPs were performed on lysates from cells described in (G) or from ego3∆ cells expressing Ego3WT-HA3, and processed as in (C). HA, human influenza hemagglutinin.

  • Fig. 4 Structural comparison of the EGOC and the Ragulator-Rag complex.

    The structure of the Ragulator-Rag complex containing both GTPase and Roadblock domains of RagA-RagC are modeled from the Ragulator-RagA(CTD)-RagC(CTD) complex (PDB code 6EHR) and the Gtr1-Gtr2 heterodimer (PDB code 3R7W). Cartoon representations of the EGOC and the Ragulator-Rag complex are shown on the right side.

  • Table 1 Summary of diffraction data collection and structure refinement statistics.

    Numbers in parentheses refer to the highest resolution shell.

    Diffraction data
    Wavelength (Å)0.9789
    Space groupP212121
    Cell parameters
      a, b, c (Å)81.68, 120.55, 323.27
    Resolution (Å)49.2–3.20 (3.38–3.20)
    Observed reflections283,819 (16,435)
    Unique reflections (I/σ(I) > 0)36,895 (1853)
    Average redundancy7.7 (8.9)
    Average I/σ(I)10.6 (1.4)
    Spherical completeness (%)68.9 (22.8)
    Ellipsoidal completeness (%)94.1 (83.3)
    Rmerge*17.3 (167.9)
    CC1/20.997 (0.461)
    Refinement and structure model
    Reflections (Fo ≥ 0σ(Fo))
      Working set33,165
      Test set1863
    Rwork/Rfree (%)23.0/28.5
    No. of protein atoms13,043
    No. of ligand/ion atoms99
    Average B factor (Å2)
      All atoms72.0
      Protein72.0
      GppNHp73.3
      Mg67.7
    RMSD
      Bond lengths (Å)0.007
      Bond angles (°)1.20
    Ramachandran plot (%)
      Favored93.4
      Allowed6.6
      Disallowed0.0

    *Rmerge = ∑hkl∑i|Ii(hkl)i−〈I(hkl)〉|/∑hkl∑iIi(hkl).

    R-factor = ||Fo|−|Fc||/|Fo|.

    Supplementary Materials

    • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/9/eaax8164/DC1

      Fig. S1. Sequence alignment of Ego1 from different species as analyzed by ESPript 3.0 (34).

      Fig. S2. Deletion of residues 98 to 121 of Ego1 does not affect the vacuolar membrane location of Ego1 nor the subsequent vacuolar recruitment of the other EGOC components.

      Fig. S3. Strains expressing Ego1 and Ego3 mutant variants that are unable to bind the Rag GTPases are sensitive to rapamycin and have reduced TORC1 activity.

      Fig. S4. Overall structure of the two EGOC molecules in the asymmetric unit.

      Fig. S5. Representative composite 2Fo-Fc omit maps (contoured at 1σ) of the EGOC (molecule A).

      Fig. S6. Structural comparison of associated and free Gtr1-Gtr2 heterodimers.

      Fig. S7. Structural comparison of the EGOC and the Ragulator-Rag complex.

      Fig. S8. Analysis of the diffraction data with the Diffraction Anisotropy Server (26).

      Table S1. Strains used in this study.

      Table S2. Plasmids used in this study.

      References (3438)

    • Supplementary Materials

      This PDF file includes:

      • Fig. S1. Sequence alignment of Ego1 from different species as analyzed by ESPript 3.0 (34).
      • Fig. S2. Deletion of residues 98 to 121 of Ego1 does not affect the vacuolar membrane location of Ego1 nor the subsequent vacuolar recruitment of the other EGOC components.
      • Fig. S3. Strains expressing Ego1 and Ego3 mutant variants that are unable to bind the Rag GTPases are sensitive to rapamycin and have reduced TORC1 activity.
      • Fig. S4. Overall structure of the two EGOC molecules in the asymmetric unit.
      • Fig. S5. Representative composite 2Fo-Fc omit maps (contoured at 1σ) of the EGOC (molecule A).
      • Fig. S6. Structural comparison of associated and free Gtr1-Gtr2 heterodimers.
      • Fig. S7. Structural comparison of the EGOC and the Ragulator-Rag complex.
      • Fig. S8. Analysis of the diffraction data with the Diffraction Anisotropy Server (26).
      • Table S1. Strains used in this study.
      • Table S2. Plasmids used in this study.
      • References (3438)

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