The role of the acyl modification, palmitoylation, in Arabidopsis stem cell regulation

Abstract

Proper control of stem cell populations is key for the development of all multicellular organisms. In Arabidopsis, stem cells are located primarily in the shoot, root and floral meristems where they undergo complex regulation. The Arabidopsis shoot and root meristems are regulated by the related WUS and WOX5 pathways, respectively. Previous studies established that these pathways share the signal transduction components POLTERGEIST (POL) and PLL1. Our latest study in Plant Cell revealed key roles for acyl modifications and lipid binding in the regulation of these two type 2C protein phosphatases. Specifically, POL and PLL1 were shown to localize to the plasma membrane in a myristioylation- and palmitoylation-dependent manner, POL and PLL1 were shown to bind to membrane lipids, and POL activity was found to be stimulated in vitro by the phospholipid PI(4)P. Here, we will discuss what is currently known in Arabidopsis and other organisms about the mechanisms of palmitoylation and provide additional evidence supporting that POL and PLL1 are palmitoylated, including describing the identification of a putative Arabidopsis palmitoyl transferase as a PLL1 interactor.

Figure 1

PLL1myr^m-GFP is mis-localized following treatment with the palmitoylation inhibitor 2-bromopalmitate. (A) The amino acid sequences of the putative N-myristoylation and palmitoylation signal peptides of POL and PLL1. The glycines and cysteines where the acyl modifications, myristoyl and palmitoyl, are attached, respectively, are shaded in gray. (B) GFP fusion protein localization with or without treatment with the palmitoylation inhibitor, 2-bromopalmitate. The PLL1-GFP, PLL1myr^m-GFP and PLL1pal^m-GFP lines are as described. Plants were treated for 6 hours with 100 µM 2-bromopalmitate (Sigma) or solvent control as described. Confocal images were obtained as described with the following modifications, staining was performed using 10 µg/mL propidium iodide in the treatment solutions and the samples were mounted on the slides in the treatment solutions. While all samples showed minor changes in cellular morphology in response to treatment with 2-bromopalmitate, only the localization of the PLL1myr^m-GFP fusion protein was dramatically altered following treatment.

Figure 2

A putative Arabidopsis palmitoyltransferase (S-acyltransferase) interacts with the N-terminus of PLL1. (A) The CytoTrap yeast two hybrid system (Stratagene) was used to screen a flower cDNA library (generated at DNA Technologies, Inc., Gaithersburg, MD) in the vector pMYR (Stratagene) for interactions with the N-terminal 258 amino acids of PLL1. The bait construct was generated by cloning the appropriate region of PLL1 (amplified with the primers GCC CGG GCG ACG TCG ACG ATG GGA AGT GGA TTC TCC TCC and AAT TAA CCG CGG CGG CCG CGA TCA TGC CAT AGC TTC AAC) into the SalI and NotI sites of pSOS (Stratagene). The bait construct was then transformed into yeast strain cdc25H(a) and positive clones were identified following mating with the meristem yeast two hybrid library which was in the strain cdc25H(α). Sequence analysis revealed that two of the thirteen N-terminal PLL1 interacting clones encoded At3g48760. At3g48760 is predicted to contain four transmembrane domains (light grey), a DPG (aspartate-proline-glycine) motif (black), a TTxE (threonine-threonine-x-glutamate) motif (black) and a DHHC (aspartate-histidine-histidine-cysteine) domain (medium grey). (B) At3g48760 contains the key residues required for the palmitoyltransferase activity of other members of the DHHC family. The DHHC domain and DPG and TTxE motifs from At3g48760 are shown aligned with the corresponding domains of the Arabidopsis palmitoyltransferase, TIP1 (At5g020350) and the S. cerevisiae palmitoyltransferase, AKR1., The consensus sequences at the bottom are from Mitchell et al. 2006.