Interaction of Yeast Rab Geranylgeranyl Transferase with Its Protein and Lipid Substrates

• Published in: Biochemistry (Easton) 2002-05, Vol.41 (21), p.6805-6816
• Main Authors: Dursina, Beatrice, Thomä, Nicolas H, Sidorovitch, Vadim, Niculae, Anca, Iakovenko, Andrei, Rak, Alexy, Albert, Stefan, Ceacareanu, Alice-Corina, Kölling, Ralf, Herrmann, Christian, Goody, Roger S, Alexandrov, Kirill
• Format: Article
• Language: English
• Subjects: Adaptor Proteins, Signal Transducing; Alkyl and Aryl Transferases - metabolism; Amino Acid Sequence; Animals; Evolution, Molecular; Fungal Proteins - metabolism; Immunoglobulins - metabolism; Mammals; Molecular Sequence Data; Polyisoprenyl Phosphates - metabolism; Protein Prenylation - physiology; Protein Processing, Post-Translational - physiology; Proteins - metabolism; rab GTP-Binding Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae Proteins; Sequence Alignment; Sequence Homology, Amino Acid; Sesquiterpenes; Substrate Specificity
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi016067w

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 α and β subunits. However, the α 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.