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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Nat Struct Mol Biol. 2015 May 4;22(6):492–498. doi: 10.1038/nsmb.3015

Figure 6. ATP-dependent cross-linking of Vps24-2 to the central pore.

Figure 6

(a) Mixed Vps4 hexamers are composed on average of five VpsCF subunit and one VpsCF 206C ΔMIT , such that VpsCF 206C ΔMIT , the only subunit competent for cross-linking, is incapable of binding Vps24-2 when it is in the monomeric form. (b) Non-reducing SDS-PAGE gel of Vps24-292C (lane 1-5) and Vps24-2136C (lane 6-10) crosslinked to Vps4 single cysteine mutants. The disulfide cross-linked Vps24-2*Vps4 complexes were detected by anti-His western blot (top panel). Input Vps4 and Vps24-2 was visualized by comassie blue staining (bottom panel). (c) Bar graphs show the relative amounts of crosslinked product formed between Vps24-2 92C and 136C mutants and Vps4 single cysteine mutant under different conditions. Error bars are the s. d. of three measurements. For normalization, the lowest mean intensity was assigned a value of 1.