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Figure 6.
Fig. 6. Nucleotide binding induces conformational change
within the N-terminal region of Vps4. (a) The ESCRT-III subunits
interact with Vps4 in the presence of ATP. ESCRT-III subunits
Vps2, Vps20, Vps24 and Snaf7 were expressed as GST-tagged fusion
proteins and bound to glutathione–agarose beads (left panel).
Purified Vps4^E233Q was loaded onto the ESCRT-III subunit-bound
matrix either in the absence (−) or presence (+) of ATP.
Proteins retained on the matrix after extensive washes were
separated on 12% SDS-PAGE gel and stained with Coomassie blue
(right panel). (b) The N-terminal domain of Vps4 interacts with
the ESCRT-III subunits. Vps2 and Vps20 were expressed as
GST-tagged fusion proteins and bound to glutathione–agarose
beads. Cell lysate containing His[8]-Vps4^1–82 or
His[8]-Vps4^1–120 was loaded onto GST-Vps2-or GST-Vps20-bound
matrix. Proteins retained on the matrix after extensive washes
were separated on the SDS-PAGE gel and detected by either
Ponceau S staining (top panel) or anti-His antibody (bottom
panel). (c) Vps4^1–120 competes with full-length Vps4 for
binding to the ESCRT-III subunit Vps2. GST-Vps2 was bound to
glutathione–agarose beads. Purified Vps4^E233Q was loaded onto
Vps2-bound matrix in the presence of ATP and increasing amounts
of bovine serum albumin or Vps4^1–120. Proteins retained on
the matrix were separated on SDS-PAGE gel and detected by
Western blotting with anti-Vps4 antibody. The amount of
Vps4^E233Q was quantified by program ImageJ and shown in a bar
diagram (the amount in the first lane was set as 100%). (d) Vps4
undergoes conformational change at the linker region upon ATP
binding. Vps4^E233Q was incubated with increasing amounts of
subtilisin at 4 °C for 30 min with different nucleotides.
Digestion products were separated on 15% SDS-PAGE, followed by
Coomassie staining.
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