Functional Analysis of Arabidopsis Postprenylation CaaX Processing Enzymes and Their Function in Subcellular Protein Targeting    

Abstract

Prenylation is a posttranslational protein modification essential for developmental processes and response to abscisic acid. Following prenylation, the three C-terminal residues are proteoliticaly removed and in turn the free carboxyl group of the isoprenyl cysteine is methylated. The proteolysis and methylation, collectively referred to as CaaX processing, are catalyzed by Ste24 endoprotease or Rce1 endoprotease and by an isoprenyl cysteine methyltransferase (ICMT). Arabidopsis (Arabidopsis thaliana) contains single STE24 and RCE1 and two ICMT homologs. Here we show that in yeast (Saccharomyces cerevisiae) AtRCE1 promoted a-mating factor secretion and membrane localization of a ROP GTPase. Furthermore, green fluorescent protein fusion proteins of AtSTE24, AtRCE1, AtICMTA, and AtICMTB are colocalized in the endoplasmic reticulum, indicating that prenylated proteins reach this compartment and that CaaX processing is likely required for subcellular targeting. AtICMTB can process yeast a-factor more efficiently than AtICMTA. Sequence and mutational analyses revealed that the higher activity AtICMTB is conferred by five residues, which are conserved between yeast Ste14p, human ICMT, and AtICMTB but not in AtICMTA. Quantitative real-time reverse transcription-polymerase chain reaction and microarray data show that AtICMTA expression is significantly lower compared to AtICMTB. AtICMTA null mutants have a wild-type phenotype, indicating that its function is redundant. However, AtICMT RNAi lines had fasciated inflorescence stems, altered phylotaxis, and developed multiple buds without stem elongation. The phenotype of the ICMT RNAi lines is similar to farnesyltransferase b-subunit mutant enhanced response to abscisic acid2 but is more subtle. Collectively, the data suggest that AtICMTB is likely the major ICMT and that methylation modulates activity of prenylated proteins.Prenylation is a posttranslational protein modification essential for the function of diverse proteins. Plant mutants lacking prenyltransferase function have pleotropic phenotypes including enlargement of the shoot apical meristem, hypersensitivity to abscisic acid (ABA), retarded growth rate, and delayed flowering (Cutler et al

Isoprenylcysteine Methylation and Demethylation Regulate Abscisic Acid Signaling inArabidopsis

“…However, a T-DNA insertion three nucleotides upstream of the TGA codon of STE14A was recently reported (Bracha-Drori et al, 2008). This mutant exhibited no detectable STE14A mRNA and no observable phenotype, suggesting that its function is either dispensable or redundant with that of STE14B.…”

“…This mutant exhibited no detectable STE14A mRNA and no observable phenotype, suggesting that its function is either dispensable or redundant with that of STE14B. Transgenic plants containing an STE14B RNA interference (RNAi) construct did not exhibit a significant decrease in STE14B mRNA abundance, but a subtle phenotype was observed (Bracha-Drori et al, 2008), suggesting that either STE14B silencing occurred at the level of translation or another gene was affected by the RNAi construct (no data were presented to demonstrate reduced ICMT protein or activity). These plants exhibited altered phylotaxis, multiple axillary buds, and fasciated stems, but they did not exhibit an ABA-hypersensitive phenotype (Bracha-Drori et al, 2008).…”

“…Transgenic plants containing an STE14B RNA interference (RNAi) construct did not exhibit a significant decrease in STE14B mRNA abundance, but a subtle phenotype was observed (Bracha-Drori et al, 2008), suggesting that either STE14B silencing occurred at the level of translation or another gene was affected by the RNAi construct (no data were presented to demonstrate reduced ICMT protein or activity). These plants exhibited altered phylotaxis, multiple axillary buds, and fasciated stems, but they did not exhibit an ABA-hypersensitive phenotype (Bracha-Drori et al, 2008). Given the ABA hypersensitivity of era1, plp, ggb, and ICME ox plants, this observation is surprising, but it is impossible to interpret without evidence of reduced ICMT activity in STE14B RNAi plants.…”

RAC/ROP GTPases and Auxin Signaling

“…Based on their amino acid sequences, RAC/ROPs have been divided into two subgroups, designated type I and type II (Winge et al, 2000). Type I RAC/ROPs terminate with a canonical CaaL motif and are attached to the membrane following prenylation in the cytoplasm by the protein geranylgeranyltransferase-I (Sorek et al, , 2011 and CaaX processing in the endoplasmic reticulum (ER) (Bracha-Drori et al, 2008). Type II RAC/ROPs terminate with a GC-CG box sequence motif and are attached to the plasma membrane by S-acylation (palmitoylation) (Lavy et al, 2002;Lavy and Yalovsky, 2006).…”

A Novel Proteinase, SNOWY COTYLEDON4, Is Required for Photosynthetic Acclimation to Higher Light Intensities in Arabidopsis

“…SCO4 is a protein whose homologs are present in higher plants, according to the data sets produced by GreenCut (Grossman et al, 2010), and, by our analysis, are distantly related to but do not have the function of the canonical CaaX-type endopeptidases, RCE1 and STE24 (Bracha et al, 2002;Bracha-Drori et al, 2008) that are found throughout eukaryotes, both plant and animal (Fig. 9).…”