title: Signals and pathways regulating nucleolar retention of novel putative nucleolar GTPase NGP-1(GNL-2).
abstract: NGP-1(GNL-2) is a putative GTPase, overexpressed in breast carcinoma and localized in the nucleolus. NGP-1 belongs to the MMR1-HSR1 family of large GTPases that are emerging as crucial coordinators of signaling cascades in different cellular compartments. The members of this family share very closely related G-domains, but the signals and pathways regulating their subcellular localization and their functional relevance remain unknown. To improve our understanding of the nuclear transport mechanism of NGP-1, we have identified two nucleolar localization signals (NoLS) that are independently shown to translocate NGP-1 as well the heterologous protein to the nucleolus. Site-specific mutagenesis and immunofluorescence studies suggest that the tandem repeats of positively charged amino acids are critical for NGP-1 NoLS function. Interestingly, amino-terminal (NGP-1(1-100)) and carboxyl-terminal (NGP-1(661-731)) signals independently interact with receptors importin-β and importin-α, respectively. This investigation, for the first time, provides evidence that the interaction of importin-α with C-terminal NoLS (NGP-1(661-731)) was able to target the heterologous protein to the nucleolar compartment. Structural modeling analysis and alanine scanning mutagenesis of conserved G-domains suggest that G4 and G5 motifs are critical for GTP binding of NGP-1 and further show that the nucleolar localization of NGP-1 is regulated by a GTP gating-mediated mechanism. In addition, our data suggest that an ongoing transcription is essential for efficient localization of NGP-1 to the nucleolus. We have observed a high level of NGP-1 expression in the mitogen-activated primary human peripheral blood mononuclear cells (hPBMC) as well as in human fetal brain-derived neural precursor cells (hNPCs) in comparison to cells undergoing differentiation. Overall, the results suggest that multiple mechanisms are involved in the localization of NGP-1 to the nucleolus for the regulation of nucleolar function in cell growth and proliferation.
authors.affiliation: Laboratory of Molecular Virology and Cell Biology, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai, India.
authors.forename: Vijaykumar
authors.initials: V
authors.lastname: Chennupati
authors.forename: Debduti
authors.initials: D
authors.lastname: Datta
authors.forename: Mallireddy Ramakrishna Subba
authors.initials: MR
authors.lastname: Rao
authors.forename: Neelima
authors.initials: N
authors.lastname: Boddapati
authors.forename: Mahesh
authors.initials: M
authors.lastname: Kayasani
authors.forename: Rajan
authors.initials: R
authors.lastname: Sankaranarayanan
authors.forename: Mamata
authors.initials: M
authors.lastname: Mishra
authors.forename: Pankaj
authors.initials: P
authors.lastname: Seth
authors.forename: Chandrasekaran
authors.initials: C
authors.lastname: Mani
authors.forename: Sundarasamy
authors.initials: S
authors.lastname: Mahalingam
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002466:Cell Nucleolus / Q000201:enzymology*; D002478:Cells, Cultured; D017931:DNA Primers; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction
chemical_list: D017931:DNA Primers; D020558:GTP Phosphohydrolases