Abstract
Posttranslational lipid modifications mediate the membrane attachment of Rab GTPases, facilitating their function in regulating intracellular vesicular trafficking. In Arabidopsis, most Rab GTPases have two C-terminal cysteines and potentially can be double-geranylgeranylated by heterodimeric Rab geranylgeranyltransferases (Rab-GGTs). Genes encoding two putative α subunits and two putative β subunits of Rab-GGTs have been annotated in the Arabidopsis thaliana genome, but little is known about Rab-GGT activity in Arabidopsis. In this study, we demonstrate that four different heterodimers can be formed between putative Arabidopsis Rab-GGT α subunits RGTA1/RGTA2 and β subunits RGTB1/RGTB2, but only RGTA1·RGTB1 and RGTA1·RGTB2 exhibit bona fide Rab-GGT activity, and they are biochemically redundant in vitro. We hypothesize that RGTA2 function might be disrupted by a 12-amino acid insertion in a conserved motif. We present evidence that Arabidopsis Rab-GGTs may have preference for prenylation of C-terminal cysteines in particular positions. We also demonstrate that Arabidopsis Rab-GGTs can not only prenylate a great variety of Rab GTPases in the presence of Rab escort protein but, unlike Rab-GGT in yeast and mammals, can also prenylate certain non-Rab GTPases independently of Rab escort protein. Our findings may help to explain some of the phenotypes of Arabidopsis protein prenyltransferase mutants.
Highlights
Small GTPases serve as molecular switches that shuttle between active GTP-bound and inactive GDP-bound forms, providing transient signals to downstream effectors [1, 2]
RGTA1/2 and RGTB1/2, Encoded by Two Pairs of Paralogous Genes, Are Putative ␣ and  Subunits of Arabidopsis Rab-GGT—Based on the annotated full-length coding sequences in the the Arabidopsis Information Resource (TAIR) database and our cDNA sequencing result, 75% of the aligned nucleotides are identical between RGTA1 (At4g24490) and RGTA2 (At5g41820), whereas 85% of the aligned nucleotides are identical between RGTB1 (At5g12210) and RGTB2 (At3g12070), suggesting that RGTA1/2 and RGTB1/2 are two pairs of paralogous genes
Both RGTB1 and RGTB2 are highly conserved with mammalian Rab-GGT  subunits (RABGGTB) in protein sequences, sharing 72 and 70% similarity to rat (Rattus norvegicus) RABGGTB, respectively, suggesting that they are paralogous putative  subunits of Arabidopsis Rab-GGT
Summary
PGGT-I ␣ subunit PLP (pluripetala) cause significant developmental defects, but the mutants are viable and fertile [20]. Mammalian and plant Rab-GGT ␣ subunits have an additional immunoglobulin (Ig)-like domain and a leucine-rich repeat (LRR) domain, both of which all known PFT/PGGT-I ␣ subunits and yeast Rab-GGT ␣ subunits lack [12, 26]. It appears that neither of these two domains is required for Rab-GGT activity [28]. The biochemical activity of the other putative subunits and possible target specificity differences among different ␣1⁄7 combinations remain unknown It is unclear whether AtREP is required for Arabidopsis RabGGT activity. Our results help partially explain the survivability of Arabidopsis mutants lacking PFT/PGGT-I activity, the lack of phenotypes in PGGT-I mutants, and the observation of partial membrane localization of PGGT-I targets in PFT/PGGT-I mutants [15]
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