ABSTRACT
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a multifunctional protein with glycolytic and non-glycolytic functions, including pro-apoptotic activity. GAPDH accumulates in the nucleus after cells are treated with genotoxic drugs, and it is present in a protein complex that binds DNA modified by thioguanine incorporation. We identified a novel CRM1-dependent nuclear export signal (NES) comprising 13 amino acids (KKVVKQASEGPLK) in the C-terminal domain of GAPDH, truncation or mutation of which abrogated CRM1 binding and caused nuclear accumulation of GAPDH. Alanine scanning of the sequence encompassing the putative NES demonstrated at least two regions important for nuclear export. Site mutagenesis of Lys259 did not affect oligomerization but impaired nuclear efflux of GAPDH, indicating that this amino acid residue is essential for proper functioning of this NES. This novel NES does not contain multiple leucine residues unlike other CRM1-interacting NES, is conserved in GAPDH from multiple species, and has sequence similarities to the export signal found in feline immunodeficiency virus Rev protein. Similar sequences (KKVV*7-13PLK) were found in two other human proteins, U5 small nuclear ribonucleoprotein, and transcription factor BT3.
Subject(s)
Active Transport, Cell Nucleus , Cell Nucleus/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Karyopherins/metabolism , Receptors, Cytoplasmic and Nuclear , Alanine/chemistry , Amino Acid Sequence , Amino Acids/chemistry , Antibodies, Monoclonal , Apoptosis , Cell Line, Tumor , Chromatography , Cytosol/metabolism , DNA/metabolism , Epitopes/chemistry , Green Fluorescent Proteins , Humans , Luminescent Proteins/metabolism , Lysine/chemistry , Microscopy, Confocal , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Nuclear Localization Signals , Peptides/chemistry , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Ribonucleoprotein, U5 Small Nuclear/chemistry , Trans-Activators/chemistry , Transfection , Exportin 1 ProteinABSTRACT
The intranuclear distribution of the transcription factor Oct-4, which is specifically expressed in totipotent mice stem and germ line cells, was studied in mouse oocytes using immunogold labeling/electron microscopy and immunofluorescence/confocal laser scanning microcopy. The localization of Oct-4 was studied in transcriptionally active (uni/bilaminar follicles) and inactive (antral follicles) oocytes. Additionally, the Oct-4 distribution was examined relative to that of the unphosphorylated form of RNA polymerase II (Pol II) and splicing factor (SC 35) in the intranuclear entities such as perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granule clusters (IGCs), Cajal bodies (CBs), and nucleolus-like bodies (NLBs). It was shown that: (i) Oct-4 is localized in PFs, IGCs, and in the dense fibrillar component (DFC) of the nucleolus at the transcriptionally active stage of the oocyte nucleus; (ii) Oct-4 present in PFs and IGCs colocalizes with Pol II and SC 35 at the transcriptionally active stage; (iii) Oct-4 accumulates in NLBs, CBs, and PGs at the inert stage of the oocyte. The results confirm the previous suggestion that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcription/processing. The colocalization of Oct-4 with Pol II in both IGCs and PFs in active oocytes (uni/bilaminar follicles) suggests that Oct-4 is intimately associated with the Pol II holoenzyme before and during transcription. The colocalization of Oct-4, Pol II, and SC 35 with coilin-containing structures such as NLBs and CBs at the inert stage (antral follicles) suggests that the latter may represent storage sites for the transcription/splicing machinery during the decline of transcription.
