Figure 2. TORC1 has little impact on the interaction between Pah1 and the Nem1-Spo7 module.

(A) Biochemical interaction between Nem1 and Pah1. Plasmid-encoded Dga1-PtA or Nem1-PtA was immunoprecipitated from extracts of Pah1-HA₃-expressing wild-type (lane 1) or nem1Δ cells (lanes 2–4), respectively, that were either grown exponentially (0 min) or treated with rapamycin (RAP) for the indicated times. Lysates (Input) and immunoprecipitates (PtA-Pulldown) were subjected to SDS-PAGE and immunoblots were probed with anti-HA or anti-IgG antibodies. WT and Δ denote wild-type and deleted version of NEM1, respectively. Numbers below the PtA-Pulldown blots indicate the relative amount of Pah1-HA₃ that bound to and was pulled down with Nem1-PtA (normalized to the samples of exponentially growing cells). (B) Biochemical interaction between Spo7 and Nem1/Pah1. Plasmid-encoded Spo7-PtA was immunoprecipitated from extracts of untreated (0 min) and rapamycin-treated (RAP; 30 min) nem1Δ spo7Δ PAH1-HA₃ cells that coexpressed plasmid-encoded Nem1-HA₃. For details see (A). (C) The interaction between Nem1 and Pah1 requires Spo7. Plasmid-encoded Nem1-PtA was immunoprecipitated from extracts of exponentially growing, Pah1-HA₃-expressing nem1Δ (lane 2) or nem1Δ spo7Δ (lane 3) cells. Pah1-HA₃-expressing wild-type cells were used as control (lane 1). Please note that loss of Spo7 consistently resulted in decreased levels of Nem1. For details see (A). WT and Δ denote wild-type and deleted version(s), respectively, of the indicated gene(s). (D) The interaction between Spo7 and Pah1 does not require Nem1. Plasmid-encoded Dga1-PtA or Spo7-PtA was immunoprecipitated from extracts of exponentially growing, Pah1-HA₃-expressing nem1Δ (lane 1) or nem1Δ spo7Δ (lanes 2 and 3) cells, which coexpressed, or not, plasmid-encoded Nem1-HA₃. Please note that our anti-HA antibodies weakly cross-react with proteins that are present in cell lysates (indicated by the asterisk), but absent in the PtA-pulldown fractions. For details see (A). WT and Δ denote wild-type and deleted version(s), respectively, of the indicated gene(s). (E) Spo7 specifically interacts with both Nem1 and Pah1, while Nem1 only interacts with Spo7, but not with Pah1, when assayed in a split-ubiquitin membrane-based yeast two-hybrid assay. Interactions were tested by monitoring either growth on plates lacking adenine (-Ade), or β-galactosidase activities (in Miller units; numbers on the right of the panels represent the means of three independent experiments performed with exponentially growing cells) of cells expressing the indicated combinations of NubG-Spo7 or NubG-Nem1 and Nem1-Cub, Pah1-Cub, Spo7-Cub, or Mon1-Cub (control).