Elsevier

Methods in Enzymology

Volume 219, 1992, Pages 362-369
Methods in Enzymology

[34] Preparation of recombinant ADP-ribosylation factor

https://doi.org/10.1016/0076-6879(92)19036-6Get rights and content

Publisher Summary

ADP-ribosylation factor (ARF) proteins are originally identified and purified based on an in vitro activity as the protein cofactor required for efficient ADP-ribosylation of G8 by cholera toxin. Subsequently, ARF is shown to be a 21-kDa GTP-binding protein that is active only in the GTP-bound form. ADP-ribosylation factor activity and immunoreactivity are found in everyeukaryotic cell tested but is absent from Escherichia coli. ADP-ribosylation factor is implicated as a critical component in the protein secretory machinery in yeast (S. cerevisiae) and mammalian cells. Disruption of the ARFl gene in yeast causes a defect in N-glycosylation, similar to that seen for Ypt1 mutants, as evidenced by the formation of incompletely glycosylated invertase. In addition, as to reconstitution of recombinant ARF proteins into more crude systems that allow studies of function is begun, it is clear that having both myristoylated and nonmyristoylated forms of the protein is useful. The methods used in obtaining both nonmyristoylated and myristoylated ARF proteins are described in the chapter.

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      Either myristoylated Arf1 or [L8K]Arf1 can be used to achieve more efficient exchange and higher concentrations of Arf1•GTP when necessary for saturation kinetics. When loading with a radiolabeled GTP, myristoylated Arf1, or [L8K]Arf1 at concentrations up to 200 μM is incubated in buffer B with up to 400 μM [α32P]GTP or [γ32P]GTP (SA = 10,000–50,000 cpm/pmol) (GTP at a 2 to 5 fold excess of Arf1) and either 3 mM dimyristoylphosphatidylcholine/0.1% (w/v) sodium cholate, pH 7.4, (described in this series, Randazzo et al., 1992) or 500 μM phospholipids in the form of LUVs for 30–60 min at 30°. MgCl2 is then added to adjust the free Mg2+ concentration to 1 mM and the reaction mixture is chilled to 4° until use.

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      This key biochemical feature allows Arf to couple the GTP–GDP conformational switch to membrane binding [63]. Arf has weak structural homology with other ras-like GTP binding proteins and has specific sequence motifs, e.g., D(V/I)GGQ instead of DTAGQ as found in the other subfamilies at the second consensus GTP-binding domain [64]. Six Arf proteins have been identified in mammals, three in yeast, 12 in A. thaliana[28,65,66], and several Arf-related proteins have been found in mammals, yeast and plants [66,67].

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