Figure 4 Δpof1 cells are sensitive to protein unfolding. The cells from the stationary phase of growth were diluted to OD600 nm = 0.2, followed by 4 serial dilutions selleck screening library of
5X. A total of 5 μL of each APR-246 clinical trial dilution were spotted on SD complete media plates containing no unfolding agent, DTT or tunicamycin. The plates were incubated at 30°C for 48 h and photographed. Figure 5 ATPase activity of Pof1p and its physical interaction with Ubc7 (ubiquitin conjugase 7) protein. (A) Hydrolysis of ATP as measured by the PiPer™ Phosphate Assay Kit (Invitrogen). (-) ATP represents the result of the reaction in the absence of ATP; (-) POF1 represents the result of the spontaneous ATP hydrolysis in the absence of Pof1p; the lane labeled “”Pof1p + ATP”" contains the complete reaction. The assays were performed at 37°C for 1 h. (B) Western blot analyses of Ubc7p using the commercial antibody Ube2G2 (Abcam). The fractions were obtained from co-immunoprecipitation assays using Pof1p polyclonal antibody from the following protein extract: WT = wild type; Δpct1 and Δpof1. The asterisk shows the Pof1p-Ubc7p complex. CE = total soluble wild type cell extract; IgG buy IPI-549 LC = IgG light chain. The arrow points Ubc7p dimer. Interestingly, using the bioinformatics tool PIPE 2 (Protein-Protein
Interaction Prediction Engine, freely available at http://pipe.cgmlab.org/), with the default cutoff of 0.06 (sensitivity = 57% and specificity = 89%), we could predict an interaction between the Kar2p ATPase and Pof1p [29]. At a lower cutoff of 0.04 (sensitivity = 70% and specificity = 83%), an interaction between Pof1p and Cdc48p was predicted, which is the ATPase present during in all types of ERAD pathways [2, 29]. As a
positive control, Pof1p and Kss1p interaction was predicted using the default cutoff of 0.06. This is in agreement with experimental data showing through transcriptome data that this mitogen-activated protein kinase (MAPK) (involved in signal transduction pathways that control filamentous growth and pheromone response) interacted with POF1 [19]. As a negative control, the ATPase from vacuole VMA10 was not predicted to be an interacting partner with Pof1p, even using a lower cutoff of 0.01 (sensitivity = 92% and specificity = 47%). To validate these in silico protein-protein interactions predictions, anti-Pof1p rabbit polyclonal antibodies were produced. The interactions between Pof1p with Doa10p and with Ubc7p, two components of the ERAD pathway, were investigated, since these complexes were previously described [22]. The physical interaction between Nas2p and Pof1p could not be investigated because there is no commercially available Nas2p antibody. Doa10p and Pof1p did not co-immunoprecipitate under the cell growth conditions tested (data not shown); however, we did observe a physical interaction between Ubc7p and Pof1p in vivo (Figure 5B).