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. Author manuscript; available in PMC: 2013 Sep 3.
Published in final edited form as: Nature. 2011 Aug 24;477(7362):61–66. doi: 10.1038/nature10362

Figure 5. ATP-dependent recycling of empty Get3 from Get1.

Figure 5

(a) Insertion of purified Get3-Sec61β targeting complex into the indicated vesicles with or without an ATP regenerating system. (b) Proteoliposomes containing rGet1/2, or rGet1/2 bound with Get3 (left panel), were tested for insertion activity of purified targeting complex in the presence or absence of ATP (right panel). (c) Purified targeting complex was tested for insertion into wild-type yRMs or those from a ΔGet3 strain, with or without ATP. (d) Closeup of the Get1c-Get3 complex (magenta and blue) modeled onto the active site of the closed, ADP•AlF4--bound Get3 dimer (grey). Steric (dashed lines) and electrostatic clashes between conserved residues in Get1 and the nucleotide $-phosphate are apparent. (e) Dissociation of Get3-Get1c, monitored by FRET, upon titration with the indicated nucleotides. Curve fits of triplicate measurements (mean +/- s.e.m.) are shown. The reaction contained 10 nM Get3(D57N) and 100 nM Get1c. (f) As in (e), but with 10 nM Get3(D57N) and 200 nM Get2c.