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. 2007 Aug;6(8):1266-77.
doi: 10.1128/EC.00024-07. Epub 2007 May 18.

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Elina Nikko  1 , Bruno André
Affiliations

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase

Elina Nikko et al. Eukaryot Cell. 2007 Aug.

Abstract

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.

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Figures

FIG. 1.
FIG. 1.
Identification of Bro1-interacting proteins by means of the mbSUS. (A) Schematic presentation of the principle of the split-ubiquitin two-hybrid assay (mbSUS) for detecting interaction between two membrane-associated proteins. The N-terminal half of ubiquitin has Ile13 mutated to Gly (NubG), thus preventing nonspecific binding to the C-terminal half of ubiquitin (Cub). When the two proteins interact, the reconstituted split-ubiquitin heterodimer is recognized by a deubiquitinating enzyme (Ubp) that cleaves and liberates the in-frame-fused PLV transcription factor. PLV can then enter the nucleus and activate reporter genes containing upstream LexA binding sites. (B) Growth tests of diploid yeast clones coexpressing various NubG- and Cub-PLV fused proteins. Expression of soluble Nub with (NubG) or without (Nub-wt) the Ile13Gly substitution were used as controls. Cells were grown for 3 days on media containing urea as a sole nitrogen source and lacking adenine and histidine (− Ade − His) in order to detect interacting pairs (right) or on the same medium with adenine and histidine (+ Ade + His) used as controls. (C) Recapitulative matrix showing the data of the experiment shown in panel A plus additional data. The gray scale represents the extent of growth after 3 days on the adenine- and histidine-free medium. s, strong; m, medium; w, weak; n, no growth. (D) Domain structures of Bro1 and Doa4. Bro1 contains a conserved N-terminal “Bro1 domain”, two central coiled-coil domains, and a conserved C-terminal PRD, mostly absent from the Bro1ΔPRD/Npi3 variant. The ubiquitin hydrolase Doa4 has two catalytically important domains, histidine and cysteine boxes, and a widely conserved ELC box.
FIG. 2.
FIG. 2.
Bro1 immunoprecipitates with the ESCRT-I complex component Vps23. Anti-HA immunoprecipitates (IP: α-HA) from lysates of cells expressing Vps23-13Myc (EN156) transformed with the empty control (pYeF1), HA-Bro1 (pEN001), or HA-Bro1ΔPRD (pEN079) plasmid were immunoblotted with anti-HA (WB: α-HA) to detect Bro1 and Bro1ΔPRD or with anti-c-myc antibodies (WB: α-c-myc) to detect Vps23. Lysate samples were collected before immunoprecipitation.
FIG. 3.
FIG. 3.
The C-terminal PRD of Bro1 mediates recruitment of Doa4 to the endosome in wild-type cells. (A) Localization of GFP-Bro1 (EN035 cells), GFP-Bro1ΔPRD/Npi3 (EN040 cells) (left, wild type), and GFP-Bro1 (pEN009) in doa4Δ (OS20-3) and doa4Δ vps4Δ (36329a) cells. The cells were visualized after a 2-h induction by galactose. (B) Anti-HA immunoprecipitates (IP: α-HA) from lysates of cells expressing Doa4-13Myc (EN155) transformed with empty control (pYeF1), HA-Bro1 (pEN001), or HA-Bro1ΔPRD (pEN079) plasmid were immunoblotted with anti-HA (WB: α-HA) to detect Bro1 and Bro1ΔPRD or with anti-c-myc antibodies (WB: α-c-myc) to detect Doa4. Lysate samples were collected before immunoprecipitation. (C) Localization of Doa4-GFP (pEN124) in wild-type (23344c), bro1Δ (27092a), snf7Δ (ME029), bro1ΔPRD/npi3 (27086c), vps4Δ (ME027), vps4Δ bro1Δ (EN079), and snf7Δ bro1Δ (EN082) cells and of Doa4W782R-GFP (pEN127) in wild-type (23344c) and vps4Δ (ME027) cells. The cells were visualized after a 3-h induction by galactose.
FIG. 4.
FIG. 4.
The proline-rich C terminus of Bro1 is required for MVB sorting of CPS, but not for that of Sna3 or Ub-Phm5. Localization of the biosynthetic cargoes GFP-CPS, Sna3-GFP, and Ub-GFP-Phm5 in wild-type (23344c), bro1Δ (27092a), bro1ΔPRD/npi3 (27086c), doa4Δ (OS20-3), and doa4W782R/npi2 (27040d) cells is shown. The cells were stained with the lipophilic dye FM4-64 in order to visualize the vacuolar membrane and class E compartments.
FIG. 5.
FIG. 5.
Bro1/Alix interacts with Rsp5/Nedd4. (A) Anti-HA immunoprecipitates (IP: α-HA) from lysates of wild-type (23344c) cells transformed with control (pYeF1), HA-Bro1 (pEN001), or HA-Bro1ΔPRD (pEN079) plasmid were immunoblotted with anti-HA (WB: α-HA) or anti-Nedd4 (WB: α-Nedd4) antibodies. Lysate samples were collected before immunoprecipitation. (B) Anti-HA immunoprecipitates from lysates of cells of the 27061b strain transformed with pKT10-Nedd4 plasmid alone (control) or together with HA-Alix plasmid (pEN187) were immunoblotted with anti-HA or anti-Nedd4 antibodies. Lysate samples were collected before immunoprecipitation. (C) Anti-c-myc immunoprecipitates (IP: α-MYC) from lysates of cells expressing Doa4-13Myc (EN155) or Vps23-13Myc (EN156) or from control cells (23344c) were immunoblotted with anti-c-myc antibodies (WB: α-c-myc) to detect Doa4 or Vps23 (top) or with anti-Nedd4 antibodies to detect Rsp5 (bottom).
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