Article Figures & Data
Figures
- Fig. 1.
Binding of clathrin TD to AP180. A, Time course of binding of TD to AP180 was monitored by SPR. Traces illustrate the SPR response, expressed in kilo response units (kRU). During the time indicated by thebar, TD was passed over surfaces to which either GST-C58 (solid line) or GST (dashed line) had been coupled covalently. B, TD binds to GST-C58 in a dose-dependent manner, whereas TD does not bind to GST at all of the concentrations that were tested. Different concentrations of TD were passed over the surfaces, and the responses were followed over time. Maximum response values are plotted as a function of TD concentration. The data points represent the mean response in two experiments. Error bars indicate ± SEM. The data were fit by a rectangular hyperbolic function.
- Fig. 2.
Identification of a repetitive element in the APs.A, A PLALIGN self-homology plot of AP180 reveals that the structure of the clathrin assembly domain of AP180 (residues 305–901) is highly repetitive. The parallellines indicate regions of similarity. B, Inspection of the LALIGN alignments revealed that the repetitive element is an ∼23 amino acid degenerate sequence, typically included on a core DLL motif. The AP180 repeats were aligned with similar repeats found in the large subunits of the AP-1 and AP-2 adaptors by Megalign. Below, Alignment of the large subunits of the AP-3 and AP-4 adaptors revealed a related repetitive element that typically included a core SLL motif. The residues surrounded byblack in the alignments are identical to a structural similarity consensus (see Materials and Methods).
- Fig. 3.
Deletion of DLL motifs reduces the ability of AP180 to assemble clathrin. A, Recombinant deletion mutants of AP180 were constructed to vary the number of DLL motifs in the clathrin assembly domain (C58). The DLL motifs within full-length AP180 are denoted by asterisks.B, Deletion of the DLL motifs gradually reduced the ability of the AP180 mutants to assemble clathrin in vitro. Colors correspond to the constructs indicated in A for B–D; the data points represent the mean of three to six independent experiments; error bars indicate ± SEM. Smooth lines indicate the Hill equation fit to the data.C, The clathrin assembly activity of the AP180 mutants was a linear function of the number of DLL motifs. The concentration of assembly protein to give half-maximal assembly (EC50) was determined from fitting the data in B to the Hill equation. The error bars indicate ± SEM. D, Clathrin assembly is proportional to the concentration of DLL motifs, because the product of the EC50 × the number of DLLs motifs was constant for each construct. The EC50 values were determined from the data in B.
- Fig. 4.
A DLL-containing peptide from the clathrin assembly domain of AP180 (AP180 pep) inhibits clathrin assembly in vitro by both AP180 and AP-2.A, AP180 pep produced a concentration-dependent inhibition of clathrin assembly by AP180. The inhibition by AP180 pep was reduced when the DLL was mutated to AAA (ΔDLL) or when the sequence of amino acids within the peptide was scrambled (Scram1 andScram2). B, Concentration-dependent inhibition of AP-2-mediated clathrin assembly by AP180 pep. The inhibition by AP180 pep was reduced when the DLL was mutated to AAA (ΔDLL). In both panels the data points represent the mean of three to four independent experiments; error bars indicate ± SEM. Smooth lines are fits of the Hill equation.
- Fig. 5.
AP180 pep inhibits neurotransmitter release.A, Recordings of presynaptic and postsynaptic responses before (Control) and after the injection of AP180 pep. AP180 pep transiently reduced transmitter release below the threshold for eliciting a postsynaptic action potential. The vertical scale bar applies to Vpost (above) andVpre (below) traces. B, Time course of the inhibition of transmitter release by AP180 pep, (injected during the time indicated by the bar), as measured by the slope of the postsynaptic potential (PSP).C, AP180 pep ΔDLL had no effect on transmitter release.
- Fig. 6.
A DLL-containing AP-2 peptide (AP2pep) inhibits clathrin assembly in vitro.A, B, Clathrin assembly by both AP-2 (A) and AP180 (B) was inhibited by AP2 pep in a concentration-dependent manner, whereas a peptide in which the DLL was mutated to AAA (ΔDLL) or in which the D and L residues were exchanged (D/L) had little effect. Mutation of the downstream PVN to AAA in AP2 pep (ΔPVN) did not reduce the efficacy of the peptide, whereas the mutation of the adjacent NLD to AAA (ΔNLD) had a moderate effect.Data points represent the mean of three to four independent experiments; error bars indicate ± SEM. Smooth lines are fits of the Hill equation. C, D, The ability of AP2 pep to inhibit clathrin assembly also was examined by negative-staining electron microscopy of clathrin cages that were assembled in the presence of AP2 pep ΔDLL (Control) or AP2 peptide (AP2 pep). The scale bar in D applies to both images.
- Fig. 7.
AP2 pep also inhibits transmitter release.A, AP2 pep reversibly inhibited transmitter release when it was injected (bar) into the squid giant presynaptic terminal. B, The inhibitory effect on transmission was lost when the DLL was mutated (AP2 pep Δ DLL).C, Inhibition of transmitter release by AP2 pep was dependent on peptide concentration. Data pointsrepresent the mean inhibition measured in two to seven experiments; error bars represent ± SEM. Smooth lines indicate rectangular hyperbola fits to the data. D, Correlation between the abilities of several AP-2 peptides to inhibit synaptic transmission in vivo and clathrin assembly in vitro by AP-2.
- Fig. 8.
Presynaptic terminals injected with AP2 pep are depleted of synaptic vesicles. Terminals injected with AP2 pep had fewer synaptic vesicles than those injected with AP2 pep ΔDLL (Control). The postsynaptic spines are indicated by S; the scale bar applies to both images.
- Fig. 9.
The DLL-containing AP2 pep inhibits synaptic vesicle endocytosis at the clathrin assembly step. A, Spatial distribution of synaptic vesicles (SVs) in terminals injected with AP2 pep or controls. Data represent the mean values and SEM from 209 active zones (AZs) analyzed from two AP2 peptide-injected terminals and 184 AZs from two control terminals. B, Relative spatial distribution of SVs, determined by dividing the mean values for the AP2 pep by the control shown in A. The dashed line indicates a 1:1 ratio and represents no difference between terminals injected with AP2 pep or control peptide. C, D, AP2 pep significantly reduced the mean number of SVs (C) and coated vesicles (CVs; D) found per AZ.E, The mean area of membrane within vesicular endosomes (Endo), SVs, CVs, and the presynaptic plasma membrane (PM) was measured for each section in control and AP2 peptide-injected terminals. F–H, Distribution of SV diameters was measured from 20 active zones each in control (F) or AP2 peptide-injected terminals (G). The smooth lines indicate Gaussian functions fit to these distributions. H, Superimposition of the two distributions (normalized to the peak) reveals no change in mean SV diameter after injection of the AP2 peptide.
- Fig. 10.
Cross-linking model for clathrin assembly by APs. Multiple clathrin binding elements (shown as spikes) exist within a single clathrin AP (top). These clathrin binding elements allow a single AP to have multivalent interactions with TDs from several clathrin molecules, thereby assembling them into a cage (middle). The inset(bottom) shows our proposed arrangement of TDs from three clathrin molecules at a vertex and illustrates the multivalent cross-linking of these clathrin molecules by APs.
Tables
- Table 1.
Inhibition of clathrin assembly and synaptic transmission by AP180 and AP-2 peptides
AP180 peptides Sequence IC50 for AP-2- mediated assembly IC50 for AP180- mediated assembly Mean inhibition of transmission AP180 pep SGGATAWGDLLGEDSLAALSS 0.4 mm 0.5 mm 57% ± 5.8 (20)1-a AP180 pep ΔDLL SGGATAWG AAAGEDSLAALSS 3.11-b 4.91-b 0 (2) Scram AP180 pep 1 SG LLDSADAEGLTAAWGLGSS ND1-c 1.81-b 6.6 ± 2.8 (5) Scram AP180 pep 2 GSSLGLASEAADGSDWLLATG ND 3.31-b ND AP-2 peptides AP2 pep QGDLLGDLLNLDLGPPVNVPQ 0.3 0.4 43 ± 6.0 (28) AP2 pep ΔPVN QGDLLGDLLNLDLGP AAAVPQ 0.2 0.4 49 ± 14 (4) AP2 pep ΔNLD QGDLLGDLLAAALGPPVNVPQ 0.7 0.9 43 ± 11 (4) AP2 pep ΔDLL QGDLLG AAANLDLGPPVNVPQ 281-b 2.51-b 4.0 ± 2.6 (7) AP2 pep D/L swap QG LDDG LDDN DLDGPPVNVPQ 141-b 501-b 0 (3)
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