Abstract
A site-directed photocross-linking approach was employed to determine components that act downstream of ADP-ribosylation factor (ARF). To this end, a photolabile phenylalanine analog was incorporated at various positions of the putative effector region of the ARF molecule. Depending on the position of incorporation, we find specific and GTP-dependent interactions of ARF with two subunits of the coatomer complex, beta-COP and gamma-COP, as well as an interaction with a cytosolic protein (approximately 185 kDa). In addition, we observe homodimer formation of ARF molecules at the Golgi membrane. These data suggest that the binding site of ARF to coatomer is at the interface of its beta- and gamma-subunits, and this is in close proximity to the second site of interaction of coatomer with the Golgi membrane, the binding site within gamma-COP for cytosolic dibasic/diphenylalanine motifs.
Highlights
COPI1-coated vesicles transport proteins and lipids between intracellular compartments along the secretory pathway [1, 2]
The following criteria were applied for selection of amino acids to be replaced with the photolabile (Tmd)Phe: (i) insertion of the photolabile residue should not change the net charge of the ADP-ribosylation factor (ARF) molecule and (ii) the amino acid to be replaced should have an optimal orientation for optimal cross-link efficiencies
With the photolabile group at a different positions within the ARF molecule, we find that ARF interacts with the ␥-subunit of coatomer
Summary
COPI1-coated vesicles transport proteins and lipids between intracellular compartments along the secretory pathway [1, 2]. Incubation of [35S]ARF-(Tmd)Phe-46 with Golgi membranes in the presence of GTP␥S and subsequent irradiation of the membrane-bound ARF resulted in three cross-link products with estimated molecular masses of between 120 and 140 kDa (Fig. 1A, lane 2).
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