The inducibly expressed GTPase localizes to the endoplasmic reticulum, independently of GTP binding.
The inducibly expressed GTPase (IGTP) is representative of a newly identified group of interferon gamma-inducible GTPases, whose functions are currently unknown. We have begun to address the cellular function of IGTP by examining its subcellular distribution and its guanine nucleotide binding status. Using immunofluorescence, electron microscopy, and subcellular fractionation, IGTP was localized predominantly to the endoplasmic reticulum of both RAW 264. 7 macrophages and C127 fibroblasts. In the immunostaining experiments, staining of discrete cytoplasmic structures on the periphery of the endoplasmic reticulum was also evident. Using polyethyleneimine-cellulose thin layer chromatography, the guanine nucleotides that complexed to immunoprecipitated IGTP, in both control and interferon gamma-stimulated cells, were 90-95% GTP and 5-10% GDP, suggesting that the protein was in an active state. A mutant IGTP protein was created that had no detectable complexed GTP, and in both subcellular fractionation and IGTP-green fluorescent protein fusion studies, this mutant also localized to the endoplasmic reticulum. These results suggested that the GTP binding status of IGTP is independent 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 processing or trafficking.
Taylor
G A
GA
ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702, USA.
Stauber
R
R
Rulong
S
S
Hudson
E
E
Pei
V
V
Pavlakis
G N
GN
Resau
J H
JH
Vande Woude
G F
GF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D004790:Enzyme Induction; D005347:Fibroblasts; D020558:GTP Phosphohydrolases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008164:Luminescent Proteins / Q000096:biosynthesis; D008264:Macrophages; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D010455:Peptides; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism
D017931:DNA Primers; D008164:Luminescent Proteins; D009842:Oligopeptides; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D006160:Guanosine Triphosphate; C085706:FLAG peptide; D020558:GTP Phosphohydrolases; C101131:Igtp protein, mouse