Roles of N-terminal fatty acid acylations in membrane compartment partitioning: Arabidopsis h-type thioredoxins as a case study

Abstract

N-terminal fatty acylations (N-myristoylation [MYR] and S-palmitoylation [PAL]) are crucial modifications affecting 2 to 4% of eukaryotic proteins. The role of these modifications is to target proteins to membranes. Predictive tools have revealed unexpected targets of these acylations in Arabidopsis thaliana and other plants. However, little is known about how N-terminal lipidation governs membrane compartmentalization of proteins in plants. We show here that h-type thioredoxins (h-TRXs) cluster in four evolutionary subgroups displaying strictly conserved N-terminal modifications. It was predicted that one subgroup undergoes only MYR and another undergoes both MYR and PAL. We used plant TRXs as a model protein family to explore the effect of MYR alone or MYR and PAL in the same family of proteins. We used a high-throughput biochemical strategy to assess MYR of specific TRXs. Moreover, various TRX-green fluorescent protein fusions revealed that MYR localized protein to the endomembrane system and that partitioning between this membrane compartment and the cytosol correlated with the catalytic efficiency of the N-myristoyltransferase acting at the N terminus of the TRXs. Generalization of these results was obtained using several randomly selected Arabidopsis proteins displaying a MYR site only. Finally, we demonstrated that a palmitoylatable Cys residue flanking the MYR site is crucial to localize proteins to micropatching zones of the plasma membrane.

Figure 1.

The h-TRXs Undergo Predicted N-Terminal Protein Modifications According to Their Phylogenetic Origin. (A) All TRXs studied here are grouped in four evolutionary clusters. The phylogenetic tree uses all available h-TRXs from the five sequenced genomes included in this work: Arabidopsis (in bold, At1, At3g51030; At2, At5g39950; At3, At5g42980; At4, At1g19730; At5, At1g45145; At7, At1g59730; At8, At1g69880; At9, At3g08710; At10, At3g56420; At-CS1, At1g11530; At-CS2, At2g40790), M. truncatula (MtA, Medtr1g028610; MtB, DQ121442; MtC, Medtr5g021080; MtD, Medtr7g008470; MtE, Medtr5g038930; MtF, Medtr5g038960; MtG, Medtr5g038910; MtH, Medtr8g005420/Medtr8g005260; MtI, Medtr8g116230; MtJ, Medtr2g010800; MtK, Medtr4g111230; MtL, Medtr2g096730), rice (OsA, Os07g08840; OsB, Os03g58630; OsC, Os07g09310; OsD, Os05g07690; OsE, Os05g40190; OsF, Os01g07376; OsG, Os04g53740), P. trichocarpa (PtA, Poptr818765; PtB, Poptr710146; PtC, Poptr219472; PtD, Poptr258873; PtE, Poptr420455; PtF, Poptr825426; PtG, Poptr819244; PtH, Poptr663332; PtI, Poptr647677; PtJ, Poptr727798), and grape (VvA, Vv04g12270; VvB, Vv18g11650; VvC, Vv14g15400; VvD, Vv01g09260; VvE, Vv08g05890; VvF, Vv00g27000; VvG, Vv00g25955; VvH, Vv19g05210). Other characterized h-TRXs are included here: Psh, Ps-TRXh4 (AY170651), from pea; Gmh, Gm-TRXh1 (AY575954), from soybean; Nah, Na-TRXh (AAY42864), from N. alata; Pch, Pc-TRXh (AF159388), from Phalaris coerulescens; and Cr1, Cr-TRXh1 (CA56850), and Cr2, Cr-TRXh2 (AAO20258), from C. reinhardtii. Sequences from some genomes are denoted with letters (A, B, C, etc.) to avoid confusion. The phylogenetic tree was constructed using the Phylogeny.fr platform (www.phylogeny.fr/). The cladogram is fully representative of the calculated phylogram. Branch support values are not displayed. (B) N-terminal sequences (30 residues) of all h-type TRXs used to create the phylogenetic tree are shown, and critical positions are highlighted (Ala-2 in green, Gly-2 in blue, and Cys in red). TermiNator predicts that the highly conserved Ala-2 in subgroup 1 (yellow box) is α-acetylated. The conserved Gly-2 in the h-TRXs of subgroup 2 (red box) or 3 (green box) is predicted to be MYRed. Most of the TRXs within subgroup 3 are predicted to be double-acylated (MYR + PAL).

Figure 2.

Specific Subcellular Localization of Subgroup 2 Full-Length h-TRXs Is Dependent on MYR of the N-Gly. Confocal scanning microscopy images of GFP protein fusions of the Arabidopsis full-length h-TRXs transiently expressed in onion epidermal cells: TRXh2-GFP ([A] and [B]), TRXh2G2A-GFP ([C] and [D]), TRXh7-GFP ([E] and [F]), TRXh7G2A-GFP ([G] and [H]), TRXh8-GFP ([I] and [J]), TRXh8G2A-GFP ([K] and [L]), and control GFP ([V] to [Y]). Two different magnification levels are displayed to allow identification of subcellular structures: whole-cell (columns 1 and 3) and cortical fraction (columns 2 and 4). C, cortical cytoplasm; N, nucleus; T, transvacuolar strands; white arrows, spots; red arrows, nuclear rings. The white bar indicates the scale.

Figure 3.

Full-Length Subgroup 2 h-TRXs Are Localized at the Endomembrane and the N-Terminal Decapeptides Contain Complete Information for Specific Localization. Confocal scanning microscopy images of onion cells co-transfected with both full-length h-TRX-GFP (TRXh2-GFP, TRXh7-GFP, TRXh8-GFP) or peptides containing the first 10 amino acids of each Arabidopsis h-TRX fused to GFP (ph2-GFP from TRXh2; ph7-GFP from TRXh7; ph8-GFP from TRXh8) or control GFP alone and an mCherry-labeled Golgi marker (Glycine max MAN1, (Nelson et al., 2007)). TRX- or peptide-GFPs are visualized in green, m-Cherry is visualized in red, and co-localization is visualized in yellow (merged column). Scale bars = 15 µm.

Figure 4.

Fractionation of TRX-GFP Transfected Cells into Membrane and Cytoplasmic Constituents Confirmed the Distribution Observed in Vivo of the Different h-TRXs of Subgroup 2. Transfected cells with peptides containing the first 10 amino acids of each Arabidopsis h-TRX fused to GFP (ph2-GFP from TRXh2, ph7-GFP from TRXh7, and ph8-GFP from TRXh8) or control GFP previously checked by confocal scanning microscopy were employed for subcellular fractionation followed by immunoblot analysis using anti-GFP or anti-H⁺ATPase antibodies. Representative immunoblots are shown. M, membrane constituent; S, cytoplasmic constituent.

Figure 5.

Different Partitioning of h-TRXs of Subgroup 2 between the ER/Golgi Compartment and the Cytoplasm Strongly Correlates with the Capacity of Each Peptide to be MYRed by the NMT Enzyme. Quantitative ratiometric analysis of TRX-GFP accumulation at the Golgi/ER endomembrane compared with cytosolic accumulation (FI, fluorescence intensity; a.u., arbitrary units) is reported for each construct on the left (black columns). Values correspond to five independent experiments, each consisting of 30 replicates originating from the measurement performed from independent images. Error bars indicate sd. The right y axis (white columns) corresponds to S values.

Figure 6.

Partitioning of Randomly Chosen Monolipidated Substrates for MYR between the Endomembrane Compartment and the Cytoplasm Strongly Correlates with the MYR S Value. Confocal microscopy images of transient expression in onion cells of peptide-GFP. Peptides contain the first 10 amino acids of the following MYRed proteins from the Arabidopsis myristoylome: ph2 from TRXh2 (control), ARFC1 (At3g22950), F2KP (At1g07110), RPS5-like (At1g62630), DEM1 (At4g33400), DEM2 (At3g19240), and RPT2a (At4g29040). Each construct was coexpressed with the mCherry-labeled Golgi marker soybean MAN1 (Nelson et al., 2007). Peptide-GFP is visualized in green, mCherry is visualized in red, and colocalization is visualized in yellow (Merged). Bars = 10 µm.

Figure 7.

Photobleaching the ER/Golgi Membrane-Bound TRXs Reveals Idiosyncratic Kinetics for Each Subgroup 2 h-TRX. (A) Quantification of the FRAP experiments for ph2-GFP showing a two-phase type of gradual and continuous recovery during the postbleaching period with an ER/Golgi ph2-GFP recovery to relatively high level. The curve was obtained by fitting FRAP data to a double exponential corresponding to the equation reported inside the graph. (B) Quantification of the FRAP experiments for ph8-GFP showing a three-phase recovery process in which an extremely rapid initial recovery (within 1 s) is followed by a still rapid recovery and then a plateau without recovery toward the initial level of fluorescence. The curve was obtained by fitting FRAP data to a single exponential corresponding to the equation reported inside the graph. (C) Quantification of the FRAP experiments for ph7-GFP showing a three-phase recovery process in which an extremely rapid initial recovery (within 1 s) is followed by a still rapid recovery and then a plateau without recovery toward the initial level of fluorescence. The curve was obtained by fitting FRAP data to a single exponential corresponding to the equation reported inside the graph. t_r 1/2, relative time required for the fluorescence intensity to reach 50% of the maximum recovery fluorescence. D, relative diffusion coefficient. I₆₀, fraction of protein able to relocalize within the bleached area during 60-s postbleaching.

Figure 8.

Subgroup 3 h-TRXs with N-Terminal MYR and PAL Lipidations Are Specially Localized to the PM with a Reduced Fraction Also Located at the ER/Golgi Compartment. (A) Confocal scanning microscopy images of the Arabidopsis full-length h-TRXs of subgroup 3 fused to GFP transiently expressed in onion cells are shown (TRXh9 and CXXS2-GFP). The effect induced also by the corresponding nonmyristoylable forms are also shown (TRXh9G2A-GFP and CXXS2G2A-GFP), which is equivalent to GFP alone. Two different magnification levels are displayed: whole-cell (columns 1 and 3) and cortical fraction (columns 2 and 4). C, cortical cytoplasm; N, nucleus; PD, plasmodesmata; T, transvacuolar strands. (B) Confocal scanning microscopy images of onion cells cotransfected with full-length TRXh9-GFP or CXXS2 and the mCherry-labeled Golgi marker soybean MAN1 (Nelson et al., 2007). TRX-GFP is visualized in green, mCherry is visualized in red, and colocalization is visualized in yellow (Merged). The GFP-only control with the same mCherry-labeled Golgi (G) marker is also shown. Experiments and image capturing were performed according to methods. The white bar indicates the scale. (C) Confocal scanning microscopy images of onion cells transfected with the first 10 or 11 amino acids of TRXh9 and CXXS2 (ph9; pCXXS2). Bars = 10 µm.

Figure 9.

Double Acylation Consistently Induces Relocalization of GFP Fusion Proteins to the PM with Clustering of the Fluorescent Protein in Specific Microdomain Structures. Transmission electron micrographs of epidermal onion cells embedded in LR White resin. The cells were transfected with ph9-GFP as previously reported for confocal experiments. Immunolabeling of GFP is visualized by gold nanoparticles (arrows). In the control image, antibody against GFP was omitted. C, cytoplasm; G, Golgi apparatus; M, mitochondria; RER, rough endoplasmic reticulum. [See online article for color version of this figure.]

Figure 10.

N-Terminal Cys Residues Are Critical in Targeting MYRed TRXs to the PM. Confocal laser scanning microscopy images of onion cells transiently transformed with several mutated versions of N-terminal peptides or full-length TRXs of subgroup 3 fused to GFP. Each GFP construct was coexpressed with the mCherry-labeled Golgi marker soybean MAN1 (Nelson et al., 2007). GFP is visualized in green, mCherry is visualized in red, and colocalization is visualized in yellow. The white bar indicates the scale. (A) Subcellular localization of full-length TRXh9C4S or ph9C4S (first 10 amino acids) fused to GFP. (B) Quantitative ratiometric analysis of ph9C4S and pCXXS2C5S accumulation at the Golgi/ER endomembrane compared with cytosolic accumulation (FI, fluorescence intensity; a.u., arbitrary units) is reported for each construct on the left (gray columns). Error bars indicate sd. The right y axis (white columns) corresponds to S values. (C) Subcellular localization of ph9G2AC4S or pCXXS2G2AC5S fused to GFP or GFP alone. (D) Subcellular localization of pCXXS2C5S (first 11 amino acids) or pCXXS2C5SC10SC11S fused to GFP.

Figure 11.

In Vitro and in Vivo Analysis of Predicted N-Terminal S-Acylation followed by DTT Cleavage of Thioester Bond. (A) Protein extracts from cells used in the confocal microscopy analysis (Figure 10) were treated with 10 or 200 mM DTT, separated by SDS-PAGE, and ph9-GFP, pCXXS2-GFP, ph9C4S-GFP, and GFP proteins were detected by immunoblots using GFP antibodies. M, membrane fractions; S, soluble fractions. (B) Confocal microscopy images of onion cells transiently transformed with ph9-GFP or pCXXS2-GFP following DTT treatment of the epidermal cells. (C) Confocal microscopy images of onion cells transiently transformed with ph9C4S-GFP or pCXXS2C5SC10SC11S-GFP following DTT treatment of the epidermal cells. Each GFP construct used in (B) and (C) was coexpressed with the mCherry-labeled Golgi marker soybean MAN1 (Nelson et al., 2007). GFP is visualized in green, mCherry is visualized in red, and colocalization is visualized in yellow. The white bar indicates the scale.