Structural basis for endosomal targeting by the Bro1 domain

Abstract

Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs.

Figure 1. Overall Structure of Bro1

(A) Electron density from MAD map contoured at 1.0 σ. (B and C) Two views of the overall structure. The N-terminal region is colored cyan; the N-terminal (non-TPR) portion of the helical solenoid is colored green; the β sheet is colored yellow; the TPR domain is colored magenta; and the C-terminal region is colored orange.

Figure 2. Homology of Bro1 Domains

(A) Structural and sequence alignment of Bro1 domains. Key to symbols: red circle, TPR pocket residue; blue circle, electropositive region; magenta circles and squares, hydrophobic patch 1 and polar rim of hydrophobic patch 1, respectively; green circles and squares, hydrophobic patch 2 and polar rim of hydrophobic patch 2, respectively; black circle, buried polar cluster residues; black squares, buried polar residues stabilizing the N- or C-terminal regions, excluding TPR pocket residues. (B) Domain structures of selected Bro1 domain proteins.

Figure 3. The TPR Domain Substructure

(A and B) Structural overlay between Bro1 (magenta) and Hop1 (yellow), with the Hsp90 pentapeptide bound to Hop1 shown in green. (C) Details of interactions between the N terminus of Bro1 and the TPR-like pocket.

Figure 4. Functional Surfaces of the Bro1 Domain

(A) Ribbon diagram of Bro1 domain shown in the same perspective as (B)–(D) for reference. (B) Molecular surface of the Bro1 domain colored according to residue property: green, hydrophobic; blue, basic; red, acidic; white, uncharged polar. (C) Bro1 domain surface colored according to residue conservation as in Figure 2A. (D) Bro1 domain surface colored according to electrostatic potential, with blue electropositive and red electronegative. (E) The same surface as in (D) is rotated by 180° about the x axis to show the opposite side of the boomerang. (F) Close-up of hydrophobic patch 1. (G) Close-up of hydrophobic patch 2.

Figure 5. The Bro1 Domain and Snf7 Form a Complex through Surface Patch 1

(A) His6-tagged Bro1 1–387 and untagged Snf7 were coexpressed in E. coli and copurify on Ni-NTA and gel filtration chromatography and visualized by SDS-PAGE and Coomassie staining. Bro1 1–387 mutants I144D and L336D in surface patch 1 express at normal levels, but coexpressed Snf7 does not copurify with them. (B) Ni-NTA agarose beads were used to isolate purified His6-tagged Snf7 or His6-tagged Vps20 that was mixed either with GST-tagged Bro1 1–387 or GST alone. (C) Glutathione-sepharose beads were used to isolate either GST-tagged Bro1 1–387 or GST alone that had been mixed with a total yeast extract.

Figure 6. The Bro1 Domain Mediates Bro1 Localization

The indicated strains were stained with FM 4–64 and examined by fluorescence and DIC microscopy. Arrowheads indicate class E compartments. Scale bars equal 2 μm.

Figure 7. The Bro1 Domain and Surface Patch 1 Are Required for Cargo Sorting

The indicated strains expressing GFP-CPS were examined by fluorescence and DIC microscopy. Scale bar equals 2 μm.