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. 1998 Apr 28;95(9):5187-92.
doi: 10.1073/pnas.95.9.5187.

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

I Stagljar  1 , C Korostensky, N Johnsson, S te Heesen
Affiliations

A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo

I Stagljar et al. Proc Natl Acad Sci U S A. .

Abstract

A detection system for interactions between membrane proteins in vivo is described. The system is based on split-ubiquitin [Johnsson, N. & Varshavsky, A. (1994) Proc. Natl. Acad. Sci. USA 91, 10340-10344]. Interaction between two membrane proteins is detected by proteolytic cleavage of a protein fusion. The cleavage releases a transcription factor, which activates reporter genes in the nucleus. As a result, interaction between membrane proteins can be analyzed by the means of a colorimetric assay. We use membrane proteins of the endoplasmic reticulum as a model system. Wbp1p and Ost1p are both subunits of the oligosaccharyl transferase membrane protein complex. The Alg5 protein also localizes to the membrane of the endoplasmic reticulum, but does not interact with the oligosaccharyltransferase. Specific interactions are detected between Wbp1p and Ost1p, but not between Wbp1p and Alg5p. The new system might be useful as a genetic and biochemical tool for the analysis of interactions between membrane proteins in vivo.

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Figures

Figure 1
Figure 1
Principle of the split-ubiquitin system. Split-ubiquitin is drawn as [NubI:Cub] or [NubG:Cub]. The liberated reporter protein is underlined. “:” indicates ubiquitin interaction; “=” indicates interaction between protein1 and protein2; for more information see the text.
Figure 2
Figure 2
Design of Wbp1-Cub-PLV fusion protein and the principle of detection of the interaction. (A) The structure of the mature Wbp1-Cub-PLV fusion protein. Cleavage by the UBP(s) occurs at the C terminus of Cub, cleaving the Wbp1-Cub-PLV fusion protein of ≈100 kDa into Wbp1-Cub (52 kDa) and PLV (47 kDa). (B) Expression of PLV as a fusion to the ER membrane protein Wbp1p prevents the transcription factor from gene activation in the nucleus. Cleavage of Wbp1-Cub-PLV by UBP does not occur (solid scissors) in the absence of Nub, the cells are white in the presence of X-Gal and are His auxotrophs. (C) Coexpression of Wbp1-Cub-PLV with the noninteracting NubG-Alg5p does not lead to formation of the split-ubiquitin heterodimer, nor cleavage by UBP (solid scissors) and gene activation. (D) Interaction between Wbp1 and Ost1 results in formation of the split-ubiquitin heterodimer. The heterodimer is recognized and cleaved by the UBP (open scissors), liberating PLV. PLV can enter the nucleus by diffusion and bind to the LexA-binding sites leading to activation of transcription of the lacZ and HIS3 reporter genes. This results in blue cells in the presence of X-Gal and growth of the cells on agar plates lacking histidine.
Figure 3
Figure 3
β-Gal activity of cells expressing Wbp1-Cub-PLV together with Nub-fusion proteins. (A) YG0673 cells expressing Wbp1-Cub-PLV and (i) NubI-Alg5, NubA-Alg5, or NubG-Alg5 from a CEN/ARS plasmid; (ii) NubI-Alg5, NubA-Alg5, or NubG-Alg5 from a 2-μm plasmid; (iii) Ost1-NubI, Ost1-NubA, or Ost1-NubG from an integrated fusion gene (no wild-type Ost1p present in the cell); (iv) Ost1-NubI, Ost1-NubA, or Ost1-NubG from a 2-μm plasmid in presence of the wild-type Ost1p. As negative control, YG0673 was transformed with the vector pRS314. Cells were grown on Whatman filters, permeabilized, and incubated in the presence of X-Gal. Expression of β-gal resulted in blue cells. (B) Quantitative β-gal assay of YG0673 cells expressing Wbp1-Cub-PLV together with the vector, low copy number pRS314(NubG-ALG5) (ARS), NubG-ALG5 (2 μm), OST1-NubG (integrated fusion gene, no wild-type OST1 present), and OST1-NubG (2 μm, wild-type OST1 present). Shown are the results of one out of three independent experiments.
Figure 4
Figure 4
Growth of cells expressing Wbp1-Cub-PLV with various Nub-fusions on agar plates lacking histidine. YG0673 cells expressing Wbp1-Cub-PLV and either NubI-Alg5, NubA-Alg5, or NubG-Alg5 from a 2-μm plasmid or expressing Ost1-NubI, Ost1-NubA, or Ost1-NubG from a 2-μm plasmid were grown to logarithmic phase, spotted in serial 10-fold dilutions on selective agar plates containing 0.2 mM CuSO4 +/− histidine and were incubated for 5 days at 30°C. YG0673 transformed with the vector pRS314 served as negative control.
Figure 5
Figure 5
Western blot analysis of cells expressing Wbp1-Cub-PLV together with Nub-fusions. YG0673 cells expressing Wbp1-Cub-PLV with the vector (lane 1); with either NubI-Alg5, NubA-Alg5, or NubG-Alg5 from a CEN/ARS plasmid (lanes 2–4); or from a 2-μm plasmid (lanes 5–7). YG0673 expressing Ost1-NubI, Ost1-NubA, or Ost1-NubG as the sole source of Ost1 in the cell (lanes 8–10, integr) or from a 2-μm plasmid in presence of wild-type Ost1p (lanes 11–13). Cells were grown to logarithmic phase in selective medium; proteins were extracted and determined by Western blot analysis and probing with peroxidase-IgG as described in Materials and Methods. ∗, Unspecific degradation product.
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