Abstract
This chapter discusses the green fluorescent protein-tagged Ras proteins for intracellular localization. On the basis of immunofluorescence and immunoelectron microscopy, Ras protein was reported to be localized at the cytosolic leaflet of the plasma membrane. Analysis of Ras localization by green fluorescent protein (GFP) technology has forced a reevaluation of long-held assumptions about Ras trafficking. It was previously assumed that Ras translocated directly from the cytosol, where it is synthesized, to the plasma membrane by virtue of CAAX processing, analysis of GFP-Ras fusion proteins revealed endomembrane trafficking en route to the plasma membrane. Since its introduction into cell biology, green fluorescent protein (GFP) has revolutionized our ability to localize proteins in tissues, cells, and organelles. The basis of this revolution is the fact that the cDNA for GFP, incorporated into an appropriate expression plasmid and transfected, is sufficient for fluorescence in virtually any cell without additional cofactors The chapter details the methods used for tagging Ras proteins with GFP and imaging the fusion protein in live cells to observe subcellular localization and membrane trafficking.
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