Mutually exclusive engagement of the β2 appendage sandwich
by either PIPKIγ661 or clathrin. A, ∼100 μg
of GST (lanes a and b) or GST-β2 appendage (lanes
c-l) immobilized on glutathione-Sepharose was incubated with rat brain
cytosol alone (lanes a-d) or cytosol supplemented with 46
μm wild type (WT; lanes e and f) or
W642A (lanes i and j) PIPKIγ-(624-661) peptide or 139
μm wild type (lanes g and h) or W642A
(lanes k and l) PIPKIγ-(624-661) peptide. After
centrifugation, aliquots of ∼1.5% of each supernatant (S) and
∼10% of each washed pellet (P) were resolved by SDS-PAGE and
either stained with Coomassie Blue or transferred to nitrocellulose. Portions
of the blots were probed with anti-clathrin heavy chain (HC) mAb
TD.1, anti-PIPKIγ mAb clone 12, anti-AP180 mAb clone 34, or
affinity-purified anti-epsin 1 antibodies. Notice that although epsin 1
binding to the β2 appendage is affected by the addition of the
PIPKIγ peptide, particularly at the highest concentration, the
sandwich-binding partners are clearly much more sensitive to the competitor.
B, ∼100 μg of GST (lanes a-d), GST-β2 appendage
(lanes e-h), or GST-β2 hinge + appendage (lanes i-l)
immobilized on glutathione-Sepharose was incubated with rat brain cytosol
alone (lanes a, b, e, f, i, and
j) or rat brain cytosol supplemented with 113 μm
PIPKIγ-(624-661) polypeptide (lanes c, d, g,
h, k, and l). After centrifugation, aliquots of
∼1.5% of each supernatant (S) and ∼10% of each washed pellet
(P) were resolved by SDS-PAGE and either stained with Coomassie Blue
or transferred to nitrocellulose. Portions of the blots were probed with
anti-clathrin heavy chain (HC) mAb TD.1, anti-PIPKIγ mAb clone
12, or anti-AP180 mAb clone 34. C, ∼100 μg of GST-β2
appendage immobilized on glutathione-Sepharose was incubated with rat brain
cytosol alone (lanes a and b) or cytosol supplemented with
113 μm wild type (lanes c and d) or W642A
(lanes e and f) PIPKIγ-(624-661) polypeptide or 113
μm wild type (lanes g and h) or W642A
(lanes i and j) PIPKIγ-(460-661) polypeptide. After
centrifugation, aliquots of ∼1.5% of each supernatant (S) and
∼10% of each washed pellet (P) were resolved by SDS-PAGE and
either stained with Coomassie Blue or transferred to nitrocellulose. Portions
of the blots were probed with anti-clathrin heavy chain (HC) mAb
TD.1, anti-PIPKIγ mAb clone 12, or anti-AP180 mAb clone 34. D,
fractions (20 μg) from a preparation of rat brain clathrin-coated vesicles
were resolved by SDS-PAGE and either stained with Coomassie Blue or
transferred to nitrocellulose. Portions of the blots were probed with
anti-clathrin light chain (LC) mAb Cl57.3, anti-μ2 subunit serum,
affinity-purified anti-HIP1 antibodies, or anti-PIPKIγ mAb clone 12.
Only the relevant portions are shown. Note the strong enrichment of clathrin,
AP-2, and HIP1 (arrowheads) but exclusion of PIPKIγ (open
arrowhead) in the coated vesicle fraction.