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. 2019 Jul 30;8(8):bio044024. doi: 10.1242/bio.044024

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© 2019. Published by The Company of Biologists Ltd

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Fig. 1. — Aip5 interacts with Bud6 and Spa2 in vivo. (A) Right panel shows the cut outs of a split-Ubiquitin analysis of 533 diploid Aip5 CRU-containing yeast strains, each co-expressing a different N_ub fusion protein. The N_ub and CRU-expressing cells were independently mated four times, spotted in quadruplets, transferred onto medium containing 5-FOA and incubated for 4 days. Growth of four colonies indicates interaction. Left panel identifies the N_ub fusions of the corresponding cut outs. N_ub-Bud6- and Nub-Spa2 expressing cells are boxed (see also Fig. S1 for the complete array). (B) Bud6 and Spa2 interact independently of each other with Aip5. Haploid yeast strains (upper panel) lacking either BUD6 (middle panel) or SPA2 (lower panel) and expressing the indicated N_ub fusions were spotted in tenfold serial dilutions starting at OD₆₀₀=1 on medium containing (right panel) or not containing (left panel) 5-FOA. Cells were incubated for 7 days. (C) Split-Ub analysis as in B but with diploid yeast cells expressing full length Aip5 or fragments of Aip5 as CRU fusions together with the indicated N_ub fusions. (D) Split-Ub assay as in B but with diploid yeast cells expressing genomically integrated Aip5_Δ1000-1125 CRU and co-expressing the indicated N_ub fusions. (E) Summary of the binding-site analysis. Spa2 binding site (red), Bud6 binding site (blue) and the predicted Grx-like domain (green) are highlighted in colors.