ULTRASTRUCTURAL-LOCALIZATION OF RAS-RELATED PROTEINS USING EPITOPE-TAGGED PLASMIDS

To determine the ultrastructural distribution of H-ras, the rho protei ns rho-A, rho-B, rho-C, and the rad protein (members of the ras GTP-bi nding protein family), we used cDNA expression plasmids in which a sho rt sequence coding for the epitope recognized by the anti c-myc monocl onal antibody 9E1.0 has been inserted at the N-terminus. Each of the e xpressed proteins has this epitope as a tag, allowing its localization by light and electron microscopy by the same antibody. After nuclear microinjection of these plasmids into MDCK or Rat 2 cells, expression of the protein (6-18 hr later) was confirmed by immunofluorescence lab eling with 9E10 imaged by confocal microscopy. For ultrastructural loc alization of these tagged proteins, a method was devised to process mi croinjected cells in situ into low-temperature resin. The proteins wer e localized on the sections using 9E10 detected with colloidal gold co njugates. Ha-ras protein was localized almost exclusively on the cell membranes. Rho-A and rho-C were predominantly associated with the subm embraneous actin network, and rho-B was found in association with mult ivesicular bodies. Rad protein induces the formation of large pinocyto tic vesicles and was detected on the cytoplasmic face of these vacuole s. These experiments demonstrate the successful use of this approach f or detection of de novo synthesized proteins from microinjected plasmi ds by both light and electron microscopy on a small (<50 cells) sample size.