Specific Prenylation of Tomato Rab Proteins by Geranylgeranyl Type-II Transferase Requires a Conserved Cysteine-Cysteine Motif

Abstract

Posttranslational isoprenylation of some small CTP-binding proteins is required for their biological activity. Rab geranylgeranyl transferase (Rab CCTase) uses geranylgeranyl pyrophosphate to modify Rab proteins, i t s only known substrates. Ceranylgeranylation of Rabs is believed t o promote their association with target membranes and interaction with other proteins. Plants, like other eukaryotes, contain Rab-like proteins that are associated with intracellular membranes. However, t o our knowledge, the geranylgeranylation of Rab proteins has not yet been characterized from any plant source. This report presents an activity assay that allows the characterization of prenylation of Rab-like proteins in vitro, by protein extracts prepared from plants. Tomato Rabl proteins and mammalian Rabl a were modified by geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. This modification required a conserved cysteine-cysteine motif. A mutant form lacking the cysteine-cysteine motif could not be modified, but inhibited the geranylgeranylation of its wild-type homolog. The tomato Rab proteins were modified in vitro by protein extract prepared from yeast, but failed to become modified when the protein extract was prepared from a yeast strain containing a mutant allele for the (Y subunit of yeast Rab CGTase (bet4 ts). These results demonstrate that plant cells, like other eukaryotes, contain Rab CCTase-like activity.

Tomato Rab1A Homologs as Molecular Tools for Studying Rab Geranylgeranyl Transferase in Plant Cells

“…Figure 4A shows that LeRablBmCC, but not BSA, reduced labeling of LeRablB. Similar results were obtained with GST::LeRablCmCC (Yalovsky et al, 1996) and LeRablAmCC (data not shown).…”

“…Second, we show that adding detergent to the Rab GGTase assay increased overall modification of recombinant LeRablB, another feature the plant activity shares in common with mammalian Rab GGTase. Based on the results presented here and in an accompanying paper (Yalovsky et al, 1996), we believe that the Rab-modifying activity under investigation is due to Rab GGTase and not GGTase I or some other nove1 protein prenyl transferase.…”

“…Previous experiments showed that GST-tagged LeRab fusion protein was modified by a Rab GGTase-like activity from plant cells (Yalovsky et al, 1996). To characterize this activity in greater detail, we decided to use His-tagged recombinant LeRab proteins, as was done in studies of mammalian Rab GGTase (Seabra et al, 1992a(Seabra et al, , 1992bAndres et al, 1993;Cremers et al, 1994), to avoid the possibility that the 26-kD GST moiety of recombinant GST::LeRab might affect the Rab GGTase enzyme's ability to interact with its protein substrate.…”

“…Rab GGTase, however, requires sequences remote from the C terminus to isoprenylate its protein substrate (Moores et al, 1991;Beranger et al, 1994;Sanford et al, 1995), reflecting the need for REP to bind and present Rab to the Rab GGTase catalytic a / p heterodimer. Purified and crude Rab GGTase is inhibited by mutant Rab proteins (RabmCC) in which the prenylation motif Cys residues have been converted to other amino acids (Andres et al, 1993;Sanford et al, 1993;Yalovsky et al, 1996). GGTase I, however, is unaffected by proteins that do not contain its prenylation motif.…”

Farnesylation[mdash]A Sticky Business

“…Conversely, farnesyl (15-C) or has not been determined. geranylgeranyl (20-C), isoprenoid prodOver the past few years, changes in the ucts of the mevalonic acid (MVA) pathway, profiles of prenylated proteins during plant are attached to C-proximal cysteine development and the growth of suspension-cultured cells have been documented (Randall et al, 1993;Morehead et al, 1995), and more extensive analyses of the potential role of protein prenyltransferases in regulating progression through the plant cell cycle have been reported (Morehead et al, 1995;Lorraine et al, 1996;Schmitt et al, 1996;Yalovsky et al, 1996).…”