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A fluorescent in vivo protein‐prenylation assay
Author(s) -
Delon Christine,
Goody Roger,
Alexandrov Kirill
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb62
Subject(s) - prenylation , rab , geranylgeranylation , in vivo , choroideremia , biology , chemistry , biochemistry , gtpase , enzyme , microbiology and biotechnology , retinal
Rab proteins are important for vesicle trafficking where they sort cargo, recruit effectors and control vesicle identity. Rab proteins must be bound to vesicle membranes to carry out these functions and they are therefore prenylated on (usually) two C‐terminal cysteins. Defects in prenylation can cause pathologies such as choroideremia, while Rab geranylgeranyl transferase inhibition appears to have therapeutic effects against cancer and osteoporosis. Therefore a method to measure prenylation in vivo would be an important research and diagnostic tool. However until now, there has been a paucity of methods to measure in vivo prenylation. We present an in vitro prenylation assay based on the incorporation of a fluorescence (NBD) labeled geranylgeranyl analogue into unprenylated Rabs from cell lysates, this can be used to measure prenylation inhibition in cells. We show that this assay can distinguish between specific and general in vivo prenylation inhibitors and it allows an estimation of the IC50 value for prenylation inhibitors. Moreover the fluorescent isoprenyl analogues can also enter the cell and be incorporated into protein. The in vitro prenylation assay can also be extended to compare prenylation of different Rabs in cells, allowing investigation into a Rab ‘prenylation hierarchy’ and into functional chemical knockouts. In summary we have developed an assay to measure prenylation in the cell. This research tool can be used to develop in vivo prenylation inhibitors and to investigate the role of Rab prenylation in disease. This research was supported by the Max Planck Institute for molecular physiology.