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.

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.

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.

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.

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.