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. 2011 Apr 15;22(8):1375–1388. doi: 10.1091/mbc.E10-06-0554

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© 2011 Yewdell et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 6: — Heh1p and Pom152p lumenal domains specifically interact. (A) Schematic showing both full-length Pom152p with NPC, TM, and lumenal (lumen) domains, in addition to the product of an allele of POM152 with two tandem HA peptides (pom152-HA) inserted into the lumenal domain. As shown at right, this allele is unable to support growth of heh1Δpom152Δ strains. An heh1Δpom152Δ strain covered with a URA3/POM152 plasmid and transformed with either a LEU2/POM152 or LEU2/pom152-HA plasmid was plated onto YPD or 5-FOA (to force the loss of URA3/POM152). Images were taken after 2 d at 30°C. (B) Schematic of lumenal domain constructs and topology in ER (gray lines are monolayers) expressed as fusions to both the N (Nub/prey/2μm/TRP1) or C terminus (Cub/bait/CEN/LEU2) of ubiquitin. Cub is fused to a cleavable LexA-VP16 transcription factor released upon Cub-Nub interaction that promotes transcription of the HIS3 gene in the two-hybrid query strain (NMY32). Positive interactions are thus assessed as growth on CSM-Leu-Trp-His. AI-Alg5 and DL-Alg5 are positive (+) and negative (–) controls for bait topology and self-activation, respectively. Numbers are amino acids. Y’s are glycosylation sites. HA is two tandem HA peptides inserted into the Pom152p lumenal domain. (C) NMY32 was simultaneously transformed with plasmids expressing the bait/Cub-fusion of pom152(171–1337) and the indicated prey/Nub fusions. Transformants were spotted in 10-fold serial dilutions on CSM-Leu-Trp plates and CSM-Leu-Trp-His plates and imaged after 3 d at 30°C.