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. 2019 Sep 23;181(3):1096–1113. doi: 10.1104/pp.19.00919

Figure 2.

Figure 2.

KC1 K+ channel interacts with VAMP721, SNAP33, and SEC11 via a common N-terminal motif, RYxxWE, at the base of its VSD. A to C, Yeast mating-based split-ubiquitin assay for interaction of the VAMP721, SNAP33, and SEC11 fusions with KC1 and with Ala substitutions of key residues at the cytosolic face of the VSD as bait with Y–Cub fusions. VAMP721, SNAP33, and SEC11 as NubG-X prey fusions and with SNAP33 and SEC11 anchored via the GPI signal peptide (Zhang et al., 2018). Positive and negative controls are included in Supplemental Figure S1. Similar results were obtained in each of three independent experiments. Growth on CSM-LTUM was used to verify the presence of both bait and prey expression. CSM-LTUMAH was used to verify Ade- and His-independent growth of the yeast diploids. The addition of 50 μm Met to CSM-LTUMAH suppressed bait expression as a test for interaction specificity. Yeast was dropped at 1.0 and 0.1 OD600 in each case. Incubation time was 24 h for CSM-LTUM plate and 72 h for CSM-LTUMAH plates. Immunoblot analysis (5 μg total protein/lane) of the haploid yeast used in mating (right), used the αHA antibody for the prey fusions and the αVP16 antibody for the bait fusions.