In eukaryotic cells Rab/Ypt GTPases represent a family of key membrane traffic controllers that associate with their targeted membranes via C-terminally conjugated geranylgeranyl groups. GDP dissociation inhibitor (GDI) is a general and essential regulator of Rab recycling that extracts prenylated Rab proteins from membranes at the end of their cycle of activity and facilitates their delivery to the donor membranes. Here, we present the structure of a complex between GDI and a doubly prenylated Rab protein. We show that one geranylgeranyl residue is deeply buried in a hydrophobic pocket formed by domain II of GDI, whereas the other lipid is more exposed to solvent and is skewed across several atoms of the first moiety. Based on structural information and biophysical measurements, we propose mechanistic and thermodynamic models for GDI and Rab escort protein-mediated interaction of RabGTPase with intracellular membranes.

Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

Small GTPases from the Rab/Ypt family regulate events of vesicular traffic in eukaryotic cells. For their activity, Rab proteins require a posttranslational modification that is conferred by Rab geranylgeranyltransferase (RabGGTase), which attaches geranylgeranyl moieties onto two cysteines of their C terminus. RabGGTase is present in both lower and higher eukaryotes in the form of heterodimers composed of alpha and beta subunits. However, the alpha subunits of RabGGTases from lower eukaryotes, including Saccharomyces cerevisiae (yRabGGTase), are half the size of the corresponding subunit of the mammalian enzyme. This difference is due to the presence of additional immunoglobulin (Ig)-like and leucine rich (LRR) domains in the mammalian transferase. To understand the possible evolutionary implications and functional consequences of structural differences between RabGGTases of higher and lower eukaryotes, we have investigated the interactions of yeast RabGGTase with its lipid and protein substrate. We have demonstrated that geranylgeranyl pyrophosphate binds to the enzyme with an affinity of ca. 40 nM, while binding of farnesyl pyrophosphate is much weaker, with a K(d) value of ca. 750 nM. This finding suggests that despite the structural difference, yRabGGTase selects its lipid substrate in a fashion similar to mammalian RabGGTase. However, unlike the mammalian enzyme, yRabGGTase binds prenylated and unprenylated Ypt1p:Mrs6p complexes with similar affinities (K(d) ca. 200 nM). Moreover, in contrast to the mammalian enzyme, phosphoisoprenoids do not influence the affinity of Mrs6p for yRabGGTase. Using an in vitro prenylation assay, we have demonstrated that yRabGGTase can prenylate Rab proteins in complex with mammalian REP-1, thus indicating that neither the LRR nor the Ig-like domains, nor the recently discovered alternative pathway of catalytic complex assembly, are essential for the catalytic activity of RabGGTase. Despite the ability to function in concert with yRabGGTase in vitro, expression of mammalian REP-1 could not complement deletion of MRS6 gene in S. cerevisiae in vivo. The implications of these findings are discussed.