Localization and subcellular distribution of cellular ras gene products in rat brain

Abstract

Localization and subcellular distribution of the cellular ras gene products (c- ras p21s) in rat brain were studied by immunofluorescence and immunoblotting using a monoclonal antibody recognizing all of Ki-, Ha- and N- ras p21s. In immunohistochemical analysis, strong immunoreactivity for ras p21s was observed in the neuropile of cerebral and cerebellar cortex. On the other hand, the immunoreactivity of the neuronal perikarya and that of white matter were weak and that of non-neuronal cells was undetectable. In subcellular fractionation analysis of cerebrum, c- ras p21s were found mostly in the particulate fractions and almost half of the particulate-bound c- ras p21s were recovered in the P 2 fraction containing myelin, synaptosomes and mitochondria, approximately one-third were in the P 3 fraction containing microsomes, and the rest were in the P 1 fraction containing nuclei and cell debris. In further fractionation of the P 2 fraction, most of c- ras p21s were associated with synaptosomal fraction. In the synaptosomal fraction, c- ras p21s were highly concentrated in the fractions rich in synaptic plasma membranes and were poorly present in the other fractions rich in synaptic vesicles, intrasynaptosomal mitochondria or postsynaptic densities. The content of c- ras p21s of the original homogenate was calculated to be 0.05% of the total protein and c- ras p21s were distributed in the fractions rich in synaptic plasma membranes with approximately 4-fold enrichment over the original homogenate. These results indicate that c- ras p21s are mainly localized in the synaptic plasma membranes and microsomes and suggest that they may participate in some specific neuronal functions at these sites.