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Figure 4.
Fig. 4.  [C]
appendage-partner recognition surface. (A) Glutathione Sepharose
(25 µl packed beads) containing either wild-type GST- [C]
appendage ( [C]) or
GST- [C]
appendage F837A, R905A, R905A-F837A, R916A, or R916A-F837A
mutants were incubated with rat brain cytosol for 60 min at
4°C. The Sepharose beads were then recovered by
centrifugation, and aliquots corresponding to 1/150 of the
supernatant (S) and 1/10 of each washed pellet (P) were resolved
by SDS/PAGE and either stained with Coomassie blue (Left) or
transferred to nitrocellulose (Right). Portions of the blots
were probed with anti-epsin, anti-eps15, anti-amphiphysin,
anti-AP180, or anti-dynamin antibodies. Immunoblots from assays
performed at low (  25 µg,
L) or high ( 100 µg,
H) GST- [C] density
are indicated on the left. In the experiment performed at high
density shown, the GST- [C] F837A
mutant was not tested, and 1/40 of each supernatant was
analyzed. The position of the markers (kDa) is indicated on the
left, and only the relevant portion of each blot is shown. (B)
Ribbon diagram (32) of the platform domain viewed from the top.
Conserved residues that make up the upper surface of the
platform are colored with invariant residues shaded magenta and
conserved residues, yellow. The extended hydrogen-bonding
network is shown as small gray balls with oxygen atoms in red,
nitrogen in blue. The three highly exposed residues that have
major consequences on partner binding when mutated to Ala are
highlighted (F837, R905, R916).
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