A GPI Signal Peptide-Anchored Split-Ubiquitin (GPS) System for Detecting Soluble Bait Protein Interactions at the Membrane

Abstract

Bait fusion proteins with a glycosyl-phosphatidylinositol signal sequence anchor enable effective split ubiquitin screening for interactions with otherwise soluble membrane proteins.

Figure 1.

The Mating Based Split-Ubiquitin System (mbSUS). A, Schematic of the split-ubiquitin system. As in conventional SUS methods, ubiquitin is split between the N-terminal half (Nub) and the C-terminal half (Cub), and the latter is fused with the transcriptional activator complex Protein A-LexA-VP16 (PLV). Mutating the isoleucine at position 13 of the wild-type Nub (NubI or NubWt) to Gly yields the NubG, which blocks spontaneous reassembly of ubiquitin. The NubG-Prey and Bait-CubPLV fusion constructs are transformed into mating strains of yeast that require differing amino-acid supplementation for growth. After mating, interaction of the two proteins expressed in the diploid yeast is tested by growth on selective media as in the standard SUS assay (Grefen et al., 2010; Zhang et al., 2015, 2017). Interaction between bait and prey reassembles a functional ubiquitin and leads to cleavage and release of the PLV complex by ubiquitin-specific proteases. Critical to this technology, the bait must be a membrane-bound protein, although the prey may be either membrane-bound or soluble. In the GPS system, the bait protein is fused at the N terminus with an Exg2 GPI signal peptide-anchor sequence, which anchors it in the membrane. B, The C-terminal GPI signal sequence of the Exg2 protein of yeast. The dibasic Arg-Lys (RK) residue motif is shown in bold, and the ω-site is underlined. C, Schematic of the pExg2Met-Dest vector used to express the bait fusion protein, including the N-terminal fusion with the GPI signal sequence (see also Supplemental Fig. S1).

Figure 2.

The GPS system is suitable for analyzing protein-protein interactions of the soluble SEC11^Δ149. A, Diploid yeast expressing ^OST4pSEC11-CubPLV or ^OST4pSEC11^Δ149-CubPLV as bait and a NubG-X fusion of SYP121 and the controls (negative, NubG; positive, NubI) as prey were spotted onto different media as indicated. Growth on CSM_−LTUM was used to verify the presence of both bait and prey vectors in the diploid yeast. Growth on CSM_−LTUMAH was used to verify interaction, and additions of different concentrations of Met were used to suppress bait expression as a test for interaction strength. Diploid yeast was dropped at 1.0 and 0.1 OD₆₀₀ in each case. Incubation time was 24 h on CSM_−LTUM and 72 h on CSM_−LTUMAH. B, Diploid yeast expressing Exg2 GPI signal peptide-fused ^Exg2pSEC11-CubPLV and ^Exg2pGPI-SEC11^Δ149-CubPLV as bait, with NubG-SYP121 fusion and the controls (negative, NubG; positive, NubI) as prey spotted onto different media as indicated. Growth was as in A. C, Immunoblot analysis of the diploid yeast carried out with commercial anti-HA antibody for the prey fusions and anti-VP16 antibody for the bait fusions. Ponceau S stains were used for blotting/loading control.

Figure 3.

SEC11^Δ149 is expressed in the membrane fraction in the GPS system. Yeast membrane fractions were prepared as described before (Rosa and Correia, 1991). Twenty milliliters of yeast containing the constructs were cultured until 0.6–0.8 OD₆₀₀ and harvested by centrifugation at 4°C. The pellets were quickly frozen (−70°C) after the addition of 5 mL of 100 mm Tricine, 5 mm EDTA, and 2 mm DTT. The samples were thawed, dispersed by vortexing with 1.5-mm-diameter glass beads, and diluted with 5 mL of 0.33 m Suc, 0.1 m Tris, 5 mm EDTA, and 2 mm DTT, adjusted to pH 8. After centrifugation for 3 min at 900g, the supernatants were decanted and centrifuged for 45 min at 40,000g at 4°C. The new supernatants were used as the nonmembrane fractions, and the pellets were resuspended in 20% glycerol, 10 mm Tris, 0.1 mm EDTA, and 0.1 mm DTT, adjusted to pH 7.5, and comprised the membrane fractions. Samples harvested directly from the yeast (total), the nonmembrane fraction, and the membrane fraction were loaded and separated by gel electrophoresis at 20 μg of protein per lane. Immunoblot analysis of the samples was carried out with commercial anti-Pma1 antibody and anti-VP16 antibody for the bait fusions. Ponceau S staining was used for blotting/loading control.