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Interaction of the endoplasmic reticulum 1,2-mannosidase Mns1p with Rer1p using the split-ubiquitin system

McGill Cancer Centre, McGill University, Montréal, Québec H3G 1Y6, Canada

View larger version (16K): [in a new window]   Fig. 1. Localization of Mns1p in rer1/pep4 by indirect immunofluorescence. SKY7.1 (rer1/pep4) cells expressing Mns1p on the multicopy plasmid pYH4 were incubated with affinity-purified anti-Mns1p antibodies (A) and a monoclonal antibody that recognizes the 60 kDa subunit of the endogenous vacuolar H+-ATPase (B) as described in materials and methods. Mns1p and H+-ATPase were detected with TRITC-conjugated (A) and CY2-conjugated (B) secondary antibodies, respectively. Nuclei were visualized with DAPI staining (C). The same cells with different labeling are showed in all three figures. (The bar represents 5 µm).

View larger version (46K): [in a new window]   Fig. 2. Localization of the Y23K97 chimeric protein by indirect immunofluorescence. The staining of SNY9-1 [pep4 (A,B,C,D,E,F)] and SNH6-1C-3.1 cells [rer1/pep4 (G,H,I,J,K,L,M,N)] overexpressing Mns1p (A,B,G,H,I), Y23K97 (C,D,J,K,L) or Kre2p (E,F,M,N) was performed with affinity-purified anti-Mns1p antibodies (A,G) or affinity-purified anti-Kre2p antibodies (C,E,J,M) followed by TRITC-conjugated secondary antibodies. In rer1/pep4 cells, double labeling with anti-Mns1p and anti-vacuolar H+-ATPase antibodies (G,H) or anti-Kre2p and anti-H+-ATPase antibodies (J,K) was performed in the same cells. Endogenous vacuolar H+-ATPase was detected using a monoclonal antibody followed with CY2-conjugated secondary antibodies (H,K). DAPI staining of the nuclei is shown (B,D,F,I,L,N). Parallel arrows in figures G,H,I and in figures E and F indicate double-labeling of the same cells. (Bar, 5 µm).

View larger version (31K): [in a new window]   Fig. 3. Schematic representation of the split-ubiquitin assay. Preparation of the constructs is described in materials and methods. Cub-PLV was linked to the C-terminal of Rer1p. NubG or NubI (not shown) was linked to the N-terminal of Mns1p, Sec12p, and Alg5p, and to the C-terminal of Ost1p. Interaction between NubG-Mns1p or NubG-Sec12p and Rer1p-Cub-PLV results in the association of NubG with Cub leading to cleavage of PLV in the cytosol and a positive ß-galactosidase assay. In contrast, the lack of interaction between Ost1p-NubG and Rer1p-Cub-PLV gives a negative ß-galactosidase assay. The topology of NubG-Alg5p is also shown. The lengths of the protein domains are not to scale.

View larger version (47K): [in a new window]   Fig. 4. ß-galactosidase filter assay of cells expressing Rer1p-Cub-PLV and the different Nub-fusion constructs. MML40 cells expressing Rer1p-Cub-PLV were transformed with plasmids carrying the different Nub-fusion proteins, as indicated on the left side of each panel. Four individual colonies were grown on Whatman III filter, permeabilized and incubated in the presence of X-Gal for two hours at 37°C, as described in the Materials and Methods. Expression of ß-galactosidase resulted in blue cells. Mns1p indicates cells expressing pRS314 Mns1p (lacking the Nub), and vector indicates cells expressing pRS314 as negative controls.

View larger version (63K): [in a new window]   Fig. 5. Detection of the PLV reporter protein by western blot analysis. MML40 cells expressing Rer1p-Cub-PLV were transformed with the plasmids carrying the different Nub fusion proteins, as indicated above each lane. Western blot analysis was performed as described in the Materials and Methods. Protein A was detected with HRP-IgG. The arrow indicates a non-specific degradation product. Lane 1 corresponds to extracts from non-transformed MML40 cells. Mns1p indicates cells expressing Mns1p lacking the Nub in pRS314, and vector indicates cells transfected with pRS314, as negative controls.

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