Clathrin Binding and Assembly Activities of Expressed Domains of the Synapse-specific Clathrin Assembly Protein AP-3

doi:10.1074/jbc.270.18.10933

Clathrin Binding and Assembly Activities of Expressed Domains of the Synapse-specific Clathrin Assembly Protein AP-3 (*)

Weilan Ye(2) and
Eileen M. Lafer(1)(§)

From the (1) Center for Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center, San Antonio, Texas 78245 and the
(2) Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

^§ To whom correspondence should be addressed:
Institute of Biotechnology, University of Texas Health Science Center, 15355 Lambda Dr., San Antonio, TX 78245
. Tel.: 210-567-7220; Fax: 210-567-7277; E-mail: lafer{at}thorin.uthscsa.edu.

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)

[Abstract] 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.

Footnotes

↵* This work was supported by NINDS Grant NS29051 (to E. M. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PAGE

polyacrylamide gel electrophoresis

GST

glutathione S-transferase

BSA

bovine serum albumin

GST-AP-3

GST fused with full-length AP-3 (isoform AS15AS108)

GST-C58 kDa

GST, fused with amino acids 305-744 of AP-3 (isoform AS15AS108)

GST-N33 kDa

GST, fused with amino acids 1-304 of AP-3 (isoform AS15AS108)

GST-C16 kDa

GST, fused with amino acids 745-901 of AP-3 (isoform AS15AS108)

GST-M42 kDa

GST, fused with amino acids 305-744 of AP-3 (isoform AS15AS108)

PMSF

phenymethylsulfonyl fluoride

MES

4-morpholineethanesulfonic acid.
↵² E. M. Lafer, W. Ye, and N. H. Tannery, manuscript in preparation.