High yield production of myristoylated Arf6 small GTPase by recombinant N-myristoyl transferase

Abstract

Small GTP-binding proteins of the Arf family (Arf GTPases) interact with multiple cellular partners and with membranes to regulate intracellular traffic and organelle structure. Understanding the underlying molecular mechanisms requires in vitro biochemical assays to test for regulations and functions. Such assays should use proteins in their cellular form, which carry a myristoyl lipid attached in N-terminus. N-myristoylation of recombinant Arf GTPases can be achieved by co-expression in E. coli with a eukaryotic N-myristoyl transferase. However, purifying myristoylated Arf GTPases is difficult and has a poor overall yield. Here we show that human Arf6 can be N-myristoylated in vitro by recombinant N-myristoyl transferases from different eukaryotic species. The catalytic efficiency depended strongly on the guanine nucleotide state and was highest for Arf6-GTP. Large-scale production of highly pure N-myristoylated Arf6 could be achieved, which was fully functional for liposome-binding and EFA6-stimulated nucleotide exchange assays. This establishes in vitro myristoylation as a novel and simple method that could be used to produce other myristoylated Arf and Arf-like GTPases for biochemical assays.

Keywords: Arf; Arf-like; myristoylation; small GTPases.

Figure 1. Model of attachment of myr-Arf6•GTP to membranes. The myristoylated N-terminal amphipatic helix of Arf6 interacts with membranes, and communicates with the nucleotide-binding site by the interswitch and switch regions (shown in black). GTP is shown in ball-and-stick. Drawn from PDB entry 2J5X.

Figure 3. In vitro production of myristoylated Arf6•GTP. (A) Nano ESI-MS analysis of Arf6 before (left) and after (right) in vitro myristoylation and ammonium sulfate fractionation. The molecular mass difference is 221 Da, accounting for the addition of a myristoyl group of 221 Da. Note that the unmyristoylated Arf6 sample used here has 100% of its N-terminal methionine cleaved, and that unmyristoylated Arf6 is not detected in the myr-Arf6 sample. (B) Migration on a 15% SDS polyacrylamide gel of purified recombinant Arf6 (2 μg) before (1) and after (2) in vitro myristoylation and ammonium sulfate precipitation. Note that myr-Arf6 migrates at a slightly lower apparent mass than unprocessed Arf6. The left and right lanes contain the molecular mass markers of the indicated sizes. Proteins are stained with Coomassie blue.

Figure 2. Kinetics analysis of N-myristoylation of Arf6 by eukaryotic NMTs. Michaelis-Menten analyses of the dependence of the initial velocity (s⁻¹) as a function of Arf6 concentration. Each data point represents the mean ± SD of three independent experiments. The kinetic parameters derived from these analyses are in Table 1.

Figure 4. In vitro myristoylated Arf6 is fully functional. (A) Interaction of myr-Arf6 (2 μM) with liposomes [1 mM, containing 34.3%PC, 14%PS, 21% PS, 0.7% PtdIns(4,5)P₂ and 30% cholesterol] assessed by a flotation assay. Unbound Arf6 is recovered in the bottom fraction, liposome-bound Arf6 in the top fraction. 100% corresponds to the intensity of the input normalized to the volume for each fraction. Myr-Arf6•GDP is mostly soluble and myr-Arf6•GTP is fully bound to membranes, as previously observed for myr-Arf6 produced by co-expression with NMT in E. coli. (B) EFA6-stimulated nucleotide exchange of myr-Arf6•GDP. Representative tryptophan fluorescence kinetics traces of GDP/GTP exchange of myr-Arf6•GDP (0.4 μM) in the absence or presence of 10 nM EFA6 (as indicated) and in the presence of 100 μM liposomes. Nucleotide exchange was triggered by the addition of 100 μM GTP, at 37°C. The kinetic traces were fit to a single exponential function and yielded the k_obs reported in the text.