title: Analysis of protein prenylation in vitro and in vivo using functionalized phosphoisoprenoids.
abstract: Post-translational modifications (PTMs) expand the number of protein isoforms in eukaryotic proteome by orders of magnitude. Protein modification with isoprenoid lipids is a common PTM affecting hundreds of proteins controlling the transport of information and materials into, through, and out of the eukaryotic cell. In this modification, a soluble phosphoisoprenoid such as farnesyl (C15) or geranylgeranyl (C20) pyrophosphate moiety is recruited by one of three protein prenyltransferases to covalently modify a C-terminal cysteine(s) in a target protein. The three mammalian prenyltransferases are farnesyltransferase (FTase), geranylgeranyltransferase type I (GGTase I), and Rab geranylgeranyl transferase (also termed geranylgeranyltransferase type II - GGTase II). In this unit, synthetic isoprenoids conjugated to either a fluorophore or biotin group are used to assay the activity of protein prenyltransferases in vitro or to affinity tag prenylatable proteins in cell lysates. These protocols and their modifications can be used to study the mechanisms of protein prenylation, identify prenylation targets, and characterize inhibitors of protein prenyltransferases in vitro and in vivo.
authors.affiliation: Max Planck Institute, Dortmund, Germany.
authors.forename: Uyen T T
authors.initials: UT
authors.lastname: Nguyen
authors.forename: Yaowen
authors.initials: Y
authors.lastname: Wu
authors.forename: Andrew
authors.initials: A
authors.lastname: Goodall
authors.forename: Kirill
authors.initials: K
authors.lastname: Alexandrov
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D002623:Chemistry Techniques, Analytical; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005057:Eukaryotic Cells; D051231:Farnesyltranstransferase / Q000378:metabolism*; D007553:Isotope Labeling; D011106:Polyisoprenyl Phosphates / Q000032:analysis* / Q000737:chemistry; D020033:Protein Isoforms / Q000032:analysis / Q000737:chemistry*; D017368:Protein Prenylation; D013729:Terpenes / Q000737:chemistry; D014166:Transferases
chemical_list: D011106:Polyisoprenyl Phosphates; D020033:Protein Isoforms; C095279:Rab geranylgeranyl transferase beta-subunit; D013729:Terpenes; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D051231:Farnesyltranstransferase; D003545:Cysteine