ABSTRACT
Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9(-/-) eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9(-/-) eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9(-/-) eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.
Subject(s)
Antigens, CD/metabolism , Membrane Glycoproteins/metabolism , Ovum/metabolism , Secretory Vesicles/metabolism , Sperm-Ovum Interactions/physiology , Spermatozoa/cytology , Animals , Female , Fertilization , Green Fluorescent Proteins/metabolism , Male , Membrane Glycoproteins/deficiency , Mice , Ovum/cytology , Ovum/ultrastructure , Recombinant Fusion Proteins/metabolism , Secretory Vesicles/ultrastructure , Tetraspanin 29ABSTRACT
LAP1s (lamina-associated polypeptide 1s) are type 2 integral membrane proteins with a single membrane-spanning region of the inner nuclear membrane. We report here on the cloning of the full-length cDNA of human LAP1B (huLAP1B) that encodes 584 amino acids. The sequence homology between the predicted rat LAP1B and huLAP1B was found to be 73.6%. A topological analysis was carried out by transiently expressing N-terminal GFP fused deletion mutants of huLAP1B in cells. The transmembrane (TM) domain (aa 346-368) is required for the localization of the nuclear and endoplasmic reticulum membrane and that the TM domain and the C-terminal half of the nucleoplasmic domain (aa 190-331) are sufficient for the proper localization of LAP1B. In contrast, the well-conserved lumenal domain of the nuclear membrane is not required for its topological function. Biochemical analysis showed that huLAP1B is retained within the nucleus via interactions of the nucleoplasmic portion with nuclear components.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/genetics , Nuclear Proteins/chemistry , Nuclear Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Cell Membrane/metabolism , Cell Nucleus/metabolism , Cloning, Molecular , Cytoskeletal Proteins , DNA, Complementary , Detergents/pharmacology , Green Fluorescent Proteins , Humans , Immunoblotting , Intracellular Membranes/metabolism , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Octoxynol/pharmacology , Protein Structure, Tertiary , Rats , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Time FactorsABSTRACT
On the basis of a hypothesis that proteins encoded by the mRNAs that are transported to and translated at the dendrites/synapses may play key roles in synaptic plasticity, this study reports on attempts to isolate mRNAs which are localizing at the dendrites/synapses from mouse cerebellar synaptosomal fractions. Among 100 pieces of dendritic mRNA candidates, 10 pieces of mRNAs were found to contain the cytoplasmic polyadenylation element (CPE)-like sequences which were contained in certain mRNAs translated in dendrites. We next examined the issue of whether the CPE-like sequence-containing mRNAs (CPERs) were localized in the synapses/dendrites by means of in situ hybridization. The findings indicate that CPER9 was actually localized at the apical dendrites of a portion of cerebral cortex layer V pyramidal cells, as well as at the proximal dendrites of some of the cerebellar Purkinje cells. CPER9 was found to encode a mouse homolog of MRG15, a nuclear protein which contains a chromodomain identified in several proteins that act as regulators of transcription. Immunohistochemistry with anti-MRG15 antibodies revealed that MRG15 was localized in dendrites as well as in the nuclei of Purkinje cells. These results suggest that MRG15 may serve as a link between synaptic activity and gene expression.
