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. 2009 Jun 26;284(26):17720-30.
doi: 10.1074/jbc.M109.005447. Epub 2009 May 5.

Molecular recognition of the palmitoylation substrate Vac8 by its palmitoyltransferase Pfa3

Marissa J Nadolski  1 , Maurine E Linder
Affiliations

Molecular recognition of the palmitoylation substrate Vac8 by its palmitoyltransferase Pfa3

Marissa J Nadolski et al. J Biol Chem. .

Abstract

Palmitoylation of the yeast vacuolar protein Vac8 is important for its role in membrane-mediated events such as vacuole fusion. It has been established both in vivo and in vitro that Vac8 is palmitoylated by the Asp-His-His-Cys (DHHC) protein Pfa3. However, the determinants of Vac8 critical for recognition by Pfa3 have yet to be elucidated. This is of particular importance because of the lack of a consensus sequence for palmitoylation. Here we show that Pfa3 was capable of palmitoylating each of the three N-terminal cysteines of Vac8 and that this reaction was most efficient when Vac8 is N-myristoylated. Additionally, when we analyzed the Src homology 4 (SH4) domain of Vac8 independent of the rest of the protein, palmitoylation by Pfa3 still occurred. However, the specificity of palmitoylation seen for the full-length protein was lost, and the SH4 domain was palmitoylated by all five of the yeast DHHC proteins tested. These data suggested that a region of the protein C-terminal to the SH4 domain was important for conferring specificity of palmitoylation. This was confirmed by use of a chimeric protein in which the SH4 domain of Vac8 was swapped for that of Meh1, another palmitoylated and N-myristoylated protein in yeast. In this case we saw specificity mimic that of wild type Vac8. Competition experiments revealed that the 11th armadillo repeat of Vac8 is an important element for recognition by Pfa3. This demonstrates that regions distant from the palmitoylated cysteines are important for recognition by DHHC proteins.

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Figures

FIGURE 1.
FIGURE 1.
Palmitoylation of nonmyristoylated Vac8 by Pfa3. Vac8 proteins were expressed in bacteria with or without Nmt1 and purified using conventional chromatography. The amount of Vac8 protein was determined as detailed under “Experimental Procedures.” Increasing amounts of Vac8 were incubated with [3H]palmitoyl-CoA and with partially purified Pfa3–6xHis-FLAG (●, myr-Vac8; ■, Vac8) or without partially purified Pfa3–6xHis-FLAG (○, myr-Vac8; □, Vac8) for 10 min. For each experiment three replicates were assayed. Two were quantitated by scintillation spectroscopy as described under “Experimental Procedures” (averaged in graph), and the third was analyzed by fluorography (inset) (3-day exposure). Data shown are from a single experiment, which is representative of three independent experiments.
FIGURE 2.
FIGURE 2.
Palmitoylation of Vac8 cysteine mutants by Pfa3. A, Vac8 N-terminal cysteine residues at positions 4, 5, and 7 were mutated in various combinations to serine residues. The remaining cysteine(s) is denoted in boldface and determines the name given to each mutant. B, WT or mutant Vac8 proteins were expressed in bacteria with Nmt1 and partially purified. The amount of myr-Vac8 protein in each preparation was determined by quantitative Western blot. Samples of 250 ng myr-Vac8 were analyzed by an anti-Myc Western blot. C, partially purified Pfa3–6xHis-FLAG was incubated with 80 nm myr-Vac8 mutants and [3H]palmitoyl-CoA for 10 min. Half of each reaction (20 μl) was quantitated by scintillation spectroscopy as described under “Experimental Procedures” (graph), and the other half was analyzed by fluorography (inset) (5-day exposure). Data are the average of three independent experiments each performed in duplicate. The error bars represent the standard deviation. The 100% values are as follows: experiment 1, 18.1 fmol/min; experiment 2, 34.6 fmol/min; experiment 3, 41.2 fmol/min. D, increasing amounts of WT or C7 Vac8 were incubated with partially purified Pfa3–6xHis-FLAG and [3H]palmitoyl-CoA for 10 min. The reactions were analyzed by fluorography (top panel, 7-day exposure; bottom panel, 1-day exposure).
FIGURE 3.
FIGURE 3.
Palmitoylation of SH4 domains. A, alignment of palmitoylated yeast SH4 domains analyzed in this study. The sites, or presumed sites, of palmitoylation are denoted in boldface. B and C, membranes expressing Akr1-FLAG, FLAG-Erf2/GST-Erf4, Pfa3-FLAG, Pfa4-FLAG, Pfa5-FLAG, or empty vector (pESC) were incubated with [3H]palmitoyl-CoA and 1 μm (B) myr-Vac8-myc-6xHIS, myr-Meh1-myc-6xHIS, or myr-Ygl108-myc-6xHIS (C) myr-Vac8[SH4]-GFP-6xHIS, myr-Vac8[SH4](CΔ)-GFP-6xHIS, myr-Meh1[SH4]-GFP-6xHIS, or myr-Meh1[SH4](CΔ)-GFP-6xHIS. Reactions were analyzed by fluorography with exposure lengths ranging from 1 to 3 days. The vector-incubated (pESC) reactions from C were also analyzed by immunoblot with GFP antibodies to ensure that the substrate was not proteolyzed during the reaction. The amount analyzed by Western blot represents 40% of that analyzed by fluorography. Asterisk denotes Erf2 autoacylation.
FIGURE 4.
FIGURE 4.
Specificity of palmitoylation by Pfa3 and Akr1. Increasing amounts of myr-Vac8-myc-6xHIS (A), myr-Meh1-myc-6xHIS (B), myr-Vac8[SH4]-GFP-6xHIS (C), or myr-Meh1[SH4]-GFP-6xHIS (D) were incubated with [3H]palmitoyl-CoA and either no enzyme (●), 10 nm Pfa3-FLAG (▲), or 10 nm Akr1-FLAG (■). Reactions were analyzed by fluorography and quantitated by densitometry with exposure lengths ranging from 8 h to 2 days. Data are the average of three independent experiments. The error bars represent the standard deviation. Representative films are shown in supplemental Fig. S3A, and 100% values are shown in supplemental Table 1.
FIGURE 5.
FIGURE 5.
Palmitoylation of Meh1[SH4]-Vac8. A, membranes expressing Akr1-FLAG, FLAG-Erf2/GST-Erf4, Pfa3-FLAG, Pfa4-FLAG, Pfa5-FLAG, or empty vector (pESC) were incubated with [3H]palmitoyl-CoA and 1 μm myr-Meh1[SH4]-Vac8-myc-6xHIS. Reactions were analyzed by fluorography with an exposure length of 2 days. B, increasing amounts of myr-Meh1[SH4]-Vac8-myc-6xHIS was incubated with [3H]palmitoyl-CoA and either no enzyme (●), 10 nm Pfa3-FLAG (▲), or 10 nm Akr1-FLAG (■). Reactions were analyzed by fluorography and quantitated by densitometry with an exposure length of 2 days. Data are the average of three independent experiments. The error bars represent the standard deviation. Representative films are shown in supplemental figure S3A, and 100% values are shown in supplemental Table 1.
FIGURE 6.
FIGURE 6.
Pfa3 recognizes Vac8 armadillo repeat 11. A, increasing amounts of myr-Vac8(CΔ)-myc (●), myr-Vac8[SH4]-GFP-6xHIS (▲), myr-Vac8[SH4](CΔ)-GFP-6xHIS (■), or myr-Gαi1-6xHIS (♦) were incubated with 0.1 μm myr-Vac8-myc-6xHis, [3H]palmitoyl-CoA, and partially purified Pfa3–6xHis-FLAG. B, diagram representing the proteins analyzed in C. C, increasing amounts of myr-Vac8(CΔ)-6xHIS (●), myr-Vac8[SH4](CΔ)-GFP-6xHIS (■), myr-Vac8[Arm1–11](CΔ)-myc-6xHIS (formula image), myr-Vac8[Arm1–10](CΔ)-myc-6xHIS (♢), or myr-Vac8[ArmΔ11](CΔ)-myc-6xHIS (□) were incubated with 0.1 μm myr-Vac8-myc-6xHis, [3H]palmitoyl-CoA and partially purified Pfa3–6xHis-FLAG. A and C, reactions were analyzed by fluorography and quantitated by densitometry with an exposure length of 4 h. Data are the average of three independent experiments. The error bars represent the standard deviation. Representative films are shown in supplemental Fig. S3, B and C, and 100% values are shown in supplemental Tables 2 and 3.
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