Visual monitoring of post-translational lipid modifications using EGFP-GTPase probes in live cells.
Modification of small GTPases by lipids is required for their proper subcellular localization and biological activity. Lipids added post-translationally include both farnesyl and geranylgeranyl isoprenoids and the fatty acid palmitate. Thus, specific small molecule inhibitors of these processes cause mislocalization of small GTPases and impair their biological activity. Common biochemical methods of determining the lipid modification status or inhibitor sensitivity of small GTPases, such as in vitro prenylation assays, SDS-PAGE mobility shifts or metabolic labeling, although highly useful in their own right, cannot distinguish differences among specific subpopulations of cells, link lipid modification status with other properties of interest, or provide spatio-temporal information. An alternative method takes advantage of the tight link between small GTPase lipid modification and subcellular localization. The innate localization pattern of the enhanced green fluorescent protein, a common epitope tag frequently used in live cell imaging, is altered by fusion to modified but not unmodified small GTPases. We describe here a technique that takes advantage of these properties to monitor post-translational modifications of these proteins in a rapid, visual manner in live cells.
Keller
Patricia J
PJ
Department of Pharmacology, University of North Carolina at Chapel Hill, NC 25799, USA.
Fiordalisi
James J
JJ
Berzat
Anastacia C
AC
Cox
Adrienne D
AD
D000818:Animals; D020558:GTP Phosphohydrolases / Q000235:genetics* / Q000378:metabolism; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D050356:Lipid Metabolism; D051379:Mice; D015335:Molecular Probes; D041681:NIH 3T3 Cells; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D020558:GTP Phosphohydrolases