5 Protein geranylgeranyltransferase type I

Abstract

This chapter discusses detection and assay of protein geranylgeranyltransferase type I (PGGT-I). The prenyl donor for the protein farnesyltransferase (PFT)-catalyzed reaction is farnesyl pyrophosphate (FPP), whereas geranylgeranyl pyrophosphate (GGPP) is the prenyl donor for PGGT-I and for Rab geranylgeranyltransferase. PFT and PGGTI transfer the prenyl group to proteins and peptides with a C-terminal CaaX motif. Rab geranylgeranyltransferase operates on Rab proteins with C-terminal CXC, CC, and CCXX motifs but does not geranylgeranylate peptides with these motifs. The most common way to assay PGGT-I is to trap protein substrate on a cellulose paper disk after it has been radiogeranylgeranylated in the presence of radiolabeled GGPP that contains 3 H or 14 C at carbon-1 and to subject the disk to scintillation counting. This assay is sensitive, simple to execute, and generally gives low levels of counts per minute (cpm) in the minus enzyme and minus peptide/protein substrate controls. The chapter compares PGGT-I with PFT X-ray structure and discusses PGGT-I inhibitors.