Cytosolic and Membrane-associated Proteins Involved in the Recruitment of AP-1 Adaptors onto the Trans-Golgi Network

doi:10.1074/jbc.271.41.25446

Cytosolic and Membrane-associated Proteins Involved in the Recruitment of AP-1 Adaptors onto the Trans-Golgi Network*

From the Department of Clinical Biochemistry, University of Cambridge, Cambridge CB2 2QR, United Kingdom

^§ To whom correspondence should be addressed. Tel.: 44-1223-330163; Fax: 44-1223-330598.

↵^‡ Present address: Division of Cell and Molecular Medicine, UCSD School of Medicine, La Jolla, CA 92093-0668.

Abstract

The AP-1 adaptor complex is recruited from the cytosol onto the trans-Golgi network membrane, where it co-assembles with clathrin into a coat that drives vesicle budding. The GTPase ARF1 has been shown to be required for AP-1 recruitment, and here we demonstrate that we can reconstitute full GTPγS-dependent recruitment of adaptors onto an enriched trans-Golgi network membrane fraction by adding purified AP-1 and recombinant myristylated ARF1, indicating that these are the only soluble proteins required for binding. To identify some of the membrane proteins involved in recruitment, we have incubated permeabilized metabolically labeled cells with cytosol under conditions that promote adaptor binding, then cross-linked the samples with 3,3′dithiobis(sulfosuccinimidylproprionate), denatured by boiling in SDS, and immunoprecipitated with antibodies against the various subunits. Under these conditions, the adaptor subunits co-precipitate not only with each other and with clathrin, but also with three novel proteins: p75, which is specifically cross-linked to γ-adaptin; p80, which is specifically cross-linked to β′-adaptin; and p60, which is specifically cross-linked to AP47. These proteins are all candidates for components of the adaptor docking site on the trans-Golgi network membrane.

Footnotes

↵* This work was supported by grants from the Medical Research Council and the Wellcome Trust. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

COP

coat protein

ER

endoplasmic reticulum

MPR

mannose 6-phosphate receptor

DTSSP

3,3′-dithiobis(sulfosuccinimidylproprionate)

ARF

ADP-ribosylation factor

TES

2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.
↵² W. Mallet and F. Brodsky, personal communication.
- Received May 16, 1996.
- Revision received July 25, 1996.