Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1995 May 5;270(18):10933-9.
doi: 10.1074/jbc.270.18.10933.

Clathrin binding and assembly activities of expressed domains of the synapse-specific clathrin assembly protein AP-3

W Ye  1 , E M Lafer
Affiliations

Clathrin binding and assembly activities of expressed domains of the synapse-specific clathrin assembly protein AP-3

W Ye et al. J Biol Chem. .

Abstract

We separately expressed the 58-kDa C-terminal, 42-kDa middle, 16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called F1-20, AP180, NP185, and pp155), and determined their clathrin binding and assembly properties. The 58-kDa C-terminal region of AP-3 is able to bind to clathrin triskelia and assemble them into a homogeneous population of clathrin cages and will also bind to preassembled clathrin cages. The 42-kDa central region of AP-3 can bind to both clathrin triskelia and to clathrin cages, but cannot assemble clathrin triskelia into clathrin cages. The 16-kDa C-terminal region of AP-3 can bind to clathrin cages, but cannot bind to clathrin triskelia or assemble clathrin triskelia into clathrin cages. The clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of AP-3 is able to bind to clathrin triskelia (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26), it does not promote their assembly into clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. CHem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled clathrin cages (Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It appears that the smallest functional unit that carries out all of the reported clathrin binding and assembly properties of AP-3, essentially as well as the full-length protein, is the 58-kDa C-terminal region.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1. The 58-kDa C-terminal, 16-kDa C-terminal, and 42-kDa central region of AP-3 all bind to clathrin cages
Clathrin cage binding assays were carried out as described under “Experimental Procedures.” 20 μm concentrations of either GST-AP-3 (lanes 1–4), GST-N33 kDa (lanes 5–8), GST-C58 kDa (lanes 9–12), GST-M42 kDa (lanes 13–16), GST-C16 kDa (lanes 17–20), or GST (lanes 21–24) was incubated in the absence (−) or presence (+) of 1.8 μm (corresponding to molarity of triskelia, 1.2 mg/ml) preassembled clathrin cages. Following a low speed spin to remove nonspecific aggregates, all samples were pelleted by ultracentrifugation at 100,000 × g. The pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining (upper panel) and Western blotting (lower panel) using an anti-GST-specific monoclonal antibody visualized by the ECL detection method. Positions of clathrin heavy chain (Cla HC). GST-AP-3. GST-C58 kDa, GST-M42 kDa, GST-N33 kDa, GST-C16 kDa, clathrin light chains (Cla LCs), GST, and molecular mass markers (MW) are indicated. Essentially identical results were obtained in three independent experiments.
Fig. 2
Fig. 2. The 58-kDa C-terminal and 42-kDa central region of AP-3 bind to clathrin triskelia
Clathrin triskelia binding assays were carried out as described under “Experimental Procedures.” Glutathione-Sepharose coupled to either GST-AP-3 (lanes 1, 2, 11, and 12), GST-C58 kDa (lanes 3, 4, 13, and 14). GST-M42 kDa (lanes 5, 6, 15, and 16), GST-C16 kDa (lanes 7, 8, 17, and 18), or GST (lanes 9, 10, 19, and 20) was incubated with an equal volume of either 0.4 mg/ml clathrin triskelia (lanes 1–10) or 0.4 mg/ml BSA (lanes 11–20). Following a 2-h incubation at 4 °C with agitation, the beads were pelleted by a 5-s microcentrifugation, and the supernatant was removed (this is the flow-through, corresponding to the unbound material, labeled as F in all odd lanes). The beads were washed six times with an equal volume of ice-cold isolation buffer. The heads were then eluted by boiling for 5 min in an equal volume of 1 × SDS sample buffer (this is the eluate, corresponding to the bound material, labeled as B in all even lanes). All of the samples were analyzed by SDS-PAGE followed by Coomassie Blue staining (upper panel) and by Western blot (lower panel) using either a clathrin heavy chain-specific monoclonal antibody (lanes 1–10) or a BSA-specific monoclonal antibody (lanes 11–20), visualized by the ECL method. Positions of all proteins are indicated using the abbreviations defined in the legend to Fig. 1. Essentially identical results were obtained in three independent experiments.
Fig. 3
Fig. 3. The 58-kDa C-terminal region of AP-3 assembles clathrin
Clathrin assembly assays were performed as described under “Experimental Procedures.” 3 μm clathrin triskelia were dialyzed overnight at 4 °C against isolation buffer with the addition of 20 μm concentrations of either GST-AP-3 (lanes 1 and 2), GST-C58 kDa (lanes 3 and 4). GST-M42 kDa (lanes 5 and 6), GST-C16 kDa (lanes 7 and 8), GST-N33 kDa (lanes 9 and 10), or GST (lanes 11 and 12). Following a low speed spin to remove nonspecific aggregates, newly assembled clathrin cages were pelleted by ultracentrifugation at 100,000 × g. The pellet (P) and supernatant (S) fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining (upper panel) and Western blotting (lower panel) using an anti-clathrin heavy chain-specific monoclonal antibody. Positions of all the proteins are indicated in the same way as in Fig. 1. Essentially identical results were obtained in three independent experiments.
Fig. 4
Fig. 4. The 58-kDa C-terminal region of AP-3 assembles clathrin as efficiently as full-length AP-3
Quantitative assembly assays were carried out as described under “Experimental Procedures.” 3 μm clathrin triskelia were dialyzed overnight at 4 °C against isolation buffer with the addition of one of the following test assembly proteins at the concentrations indicated in the figure: GST-AP-3 (closed circle), GST-C58 kDa (open circle), GST-C16 kDa (closed square), GST-M42 kDa (closed triangle), or GST (×). Each data point represents the mean from three experiments with the error bars representing the standard deviations derived from three experiments.
Fig. 5
Fig. 5. Ultrastructural analyses of the cages assembled by the 58-kDa C-terminal region of AP-3 reveals a homogeneous population of clathrin cages
The morphology of the product of the clathrin assembly assays displayed in Fig. 3 were evaluated by negative staining electron microscopy. Electron micrographs are shown of clathrin assembly reactions promoted by either GST (negative control) (A), GST-AP-3 (positive control) (B), or GST-C58 kDa (test assembly protein) (C). The distribution of cage diameters assembled by GST-AP-3 and GST-C58 kDa was indistinguishable.
Fig. 6
Fig. 6. Summary of the structure-function studies of AP-3
A clathrin triskelia binding site had been found in the 33-kDa N-terminal region (1, 3). A high affinity inositol polyphosphate binding site had also been found in the 33-kDa N-terminal region (33). The 33-kDa N-terminal region had been found to not bind to clathrin cages (3) or assemble clathrin triskelia into cages (1, 3). All data concerning the 58-kDa C-terminal region, the 42-kDa central region, and the 16-kDa C-terminal region are presented in this paper. For reference, all of the binding data are shown along with the distribution of acidic (A) and basic (B) residues across the mouse AP-3 sequence (23, 27).
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Murphy JE, Pleasure IT, Puszkin S, Prasad K, Keen JH. J Biol Chem. 1991;266:4401–4408. - PubMed
    1. Morris SA, Schroder S, Plessmann U, Weber K, Ungewickell E. EMBO J. 1993;12:667–675. - PMC - PubMed
    1. Ye W, Lafer EM. J Neurosci Res. 1995;41:15–26. - PMC - PubMed
    1. Pley U, Parham P. Crit Rev Biochem Mol Biol. 1993;28:431–464. - PubMed
    1. Kirchhausen T. Curr Opin Struct Biol. 1993;3:182–188.

Publication types

LinkOut - more resources