THE INDUCIBLY EXPRESSED GTPASE LOCALIZES TO THE ENDOPLASMIC-RETICULUM, INDEPENDENTLY OF GTP-BINDING

The inducibly expressed GTPase (IGTP) is representative of a newly ide ntified group of interferon gamma-inducible GTPases, whose functions a re currently unknown. We have begun to address the cellular function o f IGTP by examining its subcellular distribution and its guanine nucle otide binding status. Using immunofluorescence, electron microscopy, a nd subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264.7 macrophages and C227 fibroblas ts. In the immunostaining experiments, staining of discrete cytoplasmi c structures on the periphery of the endoplasmic reticulum was also ev ident. Using polyethyleneimine-cellulose thin layer chromatography, th e guanine nucleotides that complexed to immunoprecipitated IGTP, in bo th control and interferon gamma-stimulated cells, were 90-95% GTP and 5-10% GDP, suggesting that the protein was in an active state. A mutan t IGTP protein was created that had no detectable complexed GTP, and i n both subcellular fractionation and IGTP-green fluorescent protein fu sion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is indepe ndent of its capacity to localize to the endoplasmic reticulum. Given these results, we propose that IGTP is representative of a new family of endoplasmic reticulum GTPases that may be involved in protein proce ssing or trafficking.