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. 2009 Mar 1;20(5):1360–1373. doi: 10.1091/mbc.E08-05-0475

Figure 2.

Figure 2.

IST1 Interacts with VPS4A MIT. (A) Yeast two-hybrid interactions between IST1 and designated VPS4A constructs. Two-hybrid interactions (top) or cotransformation controls (bottom) are shown. Note that IST1 showed a positive two-hybrid interaction with the VPS4A MIT domain but not the VPS4A ATPase cassette (compare lanes 2 and 3 and 4). (B) IST1 has two distinct MIM elements. Top, alignment of IST1321-366 (top) with CHMP6 MIM2 and CHMP1A MIM1 (middle), and with MIM consensus sequences (bottom). Bottom, biosensor binding isotherms showing pure VPS4A MIT binding to GST-IST1 proteins captured from E. coli extracts. Estimated dissociation constants (micromolar) for VPS4A MIT binding were as follows: IST1, 1.1 (black); IST1303-366, 0.8 (red); IST1323-339, 12.4 (blue); IST1340-366, 26 (green); and IST11-189, >1500 (orange). Background binding of VPS4A MIT to control GST surfaces was negligible (data not shown). (C) Mutational analyses of IST1 MIM1 and MIM2. Biosensor binding isotherms showing purified VPS4A MIT binding to GST-IST1 proteins captured from E. coli extracts. Estimated dissociation constants (micromolar) for VPS4A MIT binding were as follows: IST1, 1.1 (black); IST1L325D, 8; IST1L328D, 16; IST1L355A, 17; IST1L362A, 9 (single mutants are in blue and purple shades); IST1L325D/L355A, 88; IST1L328D/L355A, 502; and IST1L328/L362, 432 (double mutants are in red and orange shades). (D) NMR chemical shift mapping of the IST1 MIM1 and MIM2 binding sites on VPS4A MIT. Figures show residues shifted by binding of IST1 MIM1 mapped onto space filling models of the VPS4A MIT domain. Binding modes of the structurally characterized CHMP1A MIM1 (top; green) and CHMP6 (middle; blue) elements are shown for reference. The bottom panel shows a structure-based cartoon model of the VPS4A MIT-IST1321-366 complex, with the three VPS4A MIT helices depicted in gray, and the IST1 MIM1 and MIM2 elements depicted in green and blue, respectively. (E) LIP5 binds the MIM1/MIM2 region of IST1. Wild-type (WT) Myc-IST1 (lanes 1 and 2) or Myc-IST1L328D/L355A (lanes 3 and 4) coprecipitations with empty vector controls (lanes 1 and 3) or with OSF-LIP5 (lanes 2 and 4).