Characterization of the self-palmitoylation activity of the transport protein particle component Bet3

Abstract

Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.

Fig. 1

Testing the pH dependence of palmitoylation of Bet3. a Orthogonal views of the crystal structure of human Bet3: the covalently bound fatty acid inserted into the hydrophobic tunnel, amino acids mutated in this study and the single tryptophan (W 96) used for fluorescence spectroscopy are highlighted. b, c Comparison of the self-palmitoylating activity of Bet3 mutants relative to wild-type Bet3 at different pH values. b Equal amounts of purified wild-type GST-Bet3 (WT) and GST-Bet3 mutants containing amino-acid substitutions at cysteine 68 (C68S, C68A) or in the vicinity of the palmitoylation site (R67E) were incubated with [³H]-Pal-CoA in PIPES/NaCl at pH 6.5 or Tris/NaCl buffer at pH 7.5 or 8.5 for 20 min at 30°C. Samples were then subjected to SDS-PAGE and fluorography. Shown are the resulting fluorograms and the quantification of individual bands (wt = 100%) by densitometry. c Similar amounts of purified wild-type GST-Bet3 and GST-Bet3 mutants containing amino-acid substitutions at aspartate 70 (D70N) and glutamate 73 (E73Q) were incubated with Pal-CoA as described in b. Shown are the resulting fluorograms and the quantification of individual bands (D70N = 100%) by densitometry

Fig. 2

Comparison of the self-palmitoylating activity of Bet3 mutants at different pH values. a Wild-type GST-Bet3 (WT) and R67E, C68S, C68A mutants were incubated with [³H]-Pal-CoA in PIPES/NaCl buffer at pH 5.5, 6.5, or 7.5 or in Tris/NaCl buffer at pH 7.5, 8.5, or 9.5 for 20 min at 30°C. b Comparison of the self-palmitoylating activity of wild-type GST-Bet3 and GST-Bet3 E73Q, D70N, and the double mutant E73Q–D70N at different pH values as described in a. The fluorograms were quantified by densitometry of bands, and relative palmitoylation (absorbance of bands at pH 9.5 = 100%) is plotted against pH. Note that the strength of the self-palmitoylation reaction (intensity of bands) cannot be compared between the wild-type and mutant Bet3 proteins, since individual fluorograms were exposed for different time periods

Fig. 3

Self-palmitoylation of Bet3 is retarded in mutants that are less efficiently palmitoylated at neutral pH. Purified GST-Bet3 proteins, wild-type, and mutants, as described in Fig. 1, were incubated at pH 7.5 with [³H]-Pal-CoA for 1, 2.5, 5, 7.5, 10, 15, or 30 min at 30°C prior to SDS-PAGE and fluorography. The fluorograms were quantified by densitometry of bands, and relative palmitoylation (absorbance of bands after 30 min of incubation = 100%) is plotted against the time of incubation. Note that individual fluorograms were exposed for different time periods, and, thus, intensity of bands cannot be compared between wild-type and mutant Bet3 proteins

Fig. 4

X-ray structural analysis of the Trs33B:Bet3C68A mutant. a The hydrophobic tunnel of Bet3 C68A is occupied by an elongated molecule. A prominent, unaccounted electron density is visible after building the peptide chain of Bet3 (upper panel 2F ₀–F _c map, blue at 0.9 σ contour level and F ₀–F _c map, green at 3 σ contour level). Palmitate could be modeled into the hydrophobic channel (lower panel 2F ₀–F _c map, blue at 1.0 σ contour level). b Superposition of the α2-α3 loop region of mutant Bet3 C68A (green complex presented here) and wild-type Bet3 (cyan in the Trs33A:Bet3 complex, PDB: 2C0J) which were crystallized in the same space group

Fig. 5

Long-chain acyl-CoAs inhibit self-palmitoylation of Bet3 at micromolar concentration. a Myristoyl-CoA, palmitoyl-CoA, stearoyl-CoA and 9-cis-oleoyl-CoA at the indicated final concentrations were incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C. The resulting fluorograms are shown. b Acetyl-CoA and octanoyl-CoA at the indicated final concentrations were incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C. Note that millimolar concentrations of these CoA derivatives were required to reveal a slight reduction in palmitoylation. c Myr-X-CoA (a non-hydrolyzable Myr-CoA analog with a thioether instead of a thioester linking myristate and CoA) at the indicated final concentration was incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C

Fig. 6

Fluorescence spectroscopy to analyze binding of Acyl-CoAs to Bet3. a Left panel Emission spectra of purified wild-type Bet3 between 310 and 410 nm. Excitation at 295 nm. The uppermost trace represents the intrinsic tryptophan fluorescence of Bet3 in the absence of acyl-CoA. It is successively quenched upon addition of palmitoyl-CoA which was added in steps of 10 μM (final concentration) to Bet3 (subsequent traces). No further reduction of the tryptophan fluorescence occurs at a final Pal-CoA concentration of 75 μM (undermost traces). Right panel Quantitative analysis of binding of Pal-CoA to wild-type Bet3 (described in c). Error bars indicate the standard deviation calculated from three experiments. b Left panel Emission spectra of purified Bet3: Similar to A, but acetyl-CoA was added to Bet3 in steps of 1 mM (final concentration). Note that the intrinsic tryptophan fluorescence is not completely quenched, even at a final concentration of 10 mM acetyl-CoA. Right panel Quantitative analysis of acetyl-CoA binding to wild-type Bet3 (described in c). Error bars indicate the standard deviation calculated from three experiments. c Quantitative analysis of binding of Pal-CoA to wild-type and mutant Bet3. Fitting of fluorescence intensities at the emission maximum (344 nm) were normalized [(F–F ₀)/F ₀, F ₀ fluorescence intensity without ligand] and plotted against the Pal-CoA concentration. The data points were fitted and the number of acyl-CoA bindings sites listed in Tables 2 and 3 were calculated as described in the Materials and methods section. Error bars indicate the standard deviation calculated from three experiments. Where not indicated, error bars are not larger than the symbols

Fig. 7

Testing inhibitors of the self-palmitoylation reaction. a Palmitate or 2-bromo-palmitate at the indicated final concentrations were incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C. b Lyso-phosphatidylcholine, cerulenin or tunicamycin at the indicated final concentrations were incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C. #: 1% chloroform, the solvent for the three inhibitors, was added to the sample. c The designated nucleotides at the indicated final concentrations and MgCl₂ (1 mM) were incubated with wild-type GST-Bet3 and [³H]-Pal-CoA for 15 min at 30°C

Fig. 8

Testing a Bet3 mutant with a completely blocked hydrophobic tunnel. a Detail of the Bet3 crystal structure showing the hydrophobic tunnel. The fatty acid is depicted as yellow space-filling model. The amino acids mutated in A82V-A138V to block the hydrophobic tunnel are modeled purple with their electron densities indicated. The tunnel is narrowed by the two opposing valines (V138 top, V82 bottom). b Similar amounts of purified wild-type GST-Bet3 and GST-Bet3 A82V-A138V were incubated with [³H]-Pal-CoA in PIPES/NaCl at pH 6.5 or Tris/NaCl buffer at pH 7.5 or 8.5 for 20 min at 30°C. c CD spectra of wild-type Bet3 and Bet3 A82V-A138V indicate that the secondary structure of Bet3 was not changed significantly by the mutation. d Patches of hydrophobic amino acids at the surface of Bet3 that might bind the fatty acid of Acyl-CoAs. The green area contains the amino acids L60, A61, V65, G66, V77, V81, L87, G88, I89, I106, L107, P111, G159, V160, the blue area consists of F71, I93, W96, P98, A99, G100, A145, F167, I168, I171