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. 2019 Mar 8;180(1):228–239. doi: 10.1104/pp.18.01315

Figure 3.

Figure 3.

Interaction with SEC11 depends on R20R21 of SYP121. A, Alignment of the segments for SYP121 and SYP122 within the N-terminal domain. The n1, n2, and n3 segments are indicated. B, Diploid yeast expressing SEC11-Cub as bait with NubG-X fusions of SNARE chimeras and controls (negative, NubG; positive, NubI) as prey were spotted onto different media as indicated. N-terminal chimeras were constructed by replacement of the n1, n2, and n3 segments from SYP122 in SYP121. 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 was used to verify interaction with SEC11-Cub expression suppressed. Yeast was dropped at 1 and 0.1 OD600 as indicated. Incubation time was 24 h for CSM-LTUM plate and 72 h for CSM-LTUMAH plates. Immunoblot analysis (5 μg of total protein per lane) of the haploid yeast used in mating (right) used the α-HA antibody for the SNARE fusions and the α-VP16 antibody for the SEC11 fusion. C, Diploid yeast expressing SEC11-Cub as bait with NubG-X fusions of SYP121 carrying substitutions with Ala at the n2 segment and controls (negative, NubG; positive, NubI) as prey were spotted onto different media as indicated. 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 was used to verify interaction with SEC11-Cub expression suppressed. Yeast was dropped at 1 and 0.1 OD600 as indicated. Incubation time was 24 h for CSM-LTUM plate and 72 h for CSM-LTUMAH plates. Immunoblot analysis (5 μg of total protein per lane) of the haploid yeast used in mating (right) used the α-HA antibody for the SNARE fusions and the α-VP16 antibody for the SEC11 fusion.