SUBCELLULAR-DISTRIBUTION OF THE ALPHA SUBUNIT(S) OF GI - VISUALIZATION BY IMMUNOFLUORESCENT AND IMMUNOGOLD LABELING

The subcellular distribution of the alpha-subunit(s) of G(i) has an obvious bearing on the ability of this protein to interact with receptors and targets and on its potential to serve in still unexplored capacities. In this study, we have examined the distribution of G(i-alpha) by means of light and electron microscopy. The cells employed were mouse 3T3 fibroblasts, normal rat kidney fibroblasts, rat C6 glioma cells, human umbilical vein endothelial cells, and human 293 kidney fibroblasts. By indirect immunofluorescence, two patterns of G(i-alpha) were evident. The more prominent was that associated with phase-dense, cytoplasmic structures exhibiting a tubule-like morphology. A similar distribution was noted for mitochondria, indicating attachment to a subset of microtubules. The second pattern appeared as a diffuse, particulate fluorescence associated with the plasma membrane. By immunogold labeling and electron microscopy, two populations of G(i-alpha) were again evident. In this instance, labeling of the plasma membrane was the more prominent. Gold particles were most often evenly distributed along the plasma membrane and were concentrated along microspikes. The second, less abundant population of G(i-alpha) represented the subunit (or fragments) within lysosomes. Specificity in immunolabeling was confirmed in all instances by immunotransfer blotting, the use of antibodies differing in specificities for epitopes within G(i-alpha), the absence of labeling with preimmune sera, and the decrease in labeling after preincubation of antisera with appropriate peptides. These results support the proposal that several populations of G(i-alpha) exist: those evident within the cytoplasm by immunofluorescence, those present at the plasma membrane, and those evident within lysosomes by immunogold labeling.