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. 2011 Aug 1;22(15):2702–2715. doi: 10.1091/mbc.E11-02-0115

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© 2011 Roth 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 2: — Ura3-Yck2 fusion proteins delineate MPD. Ura3-Yck2 constructs, derived by fusing the indicated C-terminal portions of Yck2 to the C terminus of N-terminally FLAG/HA-tagged Ura3, were expressed from single-copy CEN/ARS plasmids via a 2-h, galactose-induced expression period. (A) Ura3-Yck2 fusion protein schematic. The Yck2 portion of each fusion protein is shown, with amino acid coordinates of the added Yck2 indicated at right. (B) IIF microscopy of Ura3-Yck2 fusion proteins. Fusion proteins were detected via their N-terminal HA epitope tag, using anti HA.11 mAb. The amino acid coordinates of the attached Yck2 portion for each fusion are indicated. Top, the localizations of GAL1_P-driven copies of the Ura3 and Yck2 parental proteins. (C) Ura3-Yck2 palmitoylation. Denatured protein extracts prepared from cells expressing the indicated Ura3-Yck2 fusions were subjected to ABE, which replaces thioester-linked acyl modifications with biotin (see Materials and Methods). Subsequently, fusion proteins were immunoprecipitated using anti-FLAG mAb and then subjected to Western analysis either with anti-biotin antibody or with anti-HA.11 mAb. (D) Effect of various internal in-frame deletions on the MPD-driven palmitoylation of the Ura3-Yck2(505-546) fusion. Left, the positions of the different MPD deletions are indicated. Right, the ABE analysis of these constructs is shown.