ABSTRACT
Fusion between membranes is mediated by specific SNARE complexes. Here we report that fibroblasts survive the absence of the trans-Golgi network/early endosomal SNARE vti1a and the late endosomal SNARE vti1b with intact organelle morphology and minor trafficking defects. Because vti1a and vti1b are the only members of their SNARE subclass and the yeast homolog Vti1p is essential for cell survival, these data suggest that more distantly related SNAREs acquired the ability to function in endosomal traffic during evolution. However, absence of vti1a and vti1b resulted in perinatal lethality. Major axon tracts were missing, reduced in size, or misrouted in Vti1a(-/-) Vti1b(-/-) embryos. Progressive neurodegeneration was observed in most Vti1a(-/-) Vti1b(-/-) peripheral ganglia. Neurons were reduced by more than 95% in Vti1a(-/-) Vti1b(-/-) dorsal root and geniculate ganglia at embryonic day 18.5. These data suggest that special demands for endosomal membrane traffic could not be met in Vti1a(-/-) Vti1b(-/-) neurons. Vti1a(-/-) and Vti1b(-/-) single deficient mice were viable without these neuronal defects, indicating that they can substitute for each other in these processes.
Subject(s)
Endosomes/metabolism , Neurogenesis/physiology , Neurons/metabolism , Qb-SNARE Proteins/metabolism , Animals , Biological Transport/physiology , Cell Line, Transformed , Endosomes/genetics , Mice , Mice, Knockout , Neurons/cytology , Qb-SNARE Proteins/geneticsABSTRACT
SNARE (soluble-N-ethylmaleimide-sensitive factor attachment receptor) proteins mediate the recognition and fusion of transport vesicles in eukaryotic cells. The SNARE protein VAMP8 (also called endobrevin) is involved in the fusion of late endosomes and in some pathways of regulated exocytosis. In a subset of mice deficient for the SNARE protein VAMP8, a severe alteration of the thymus and in T lymphocyte development was observed and characterized. The size of the thymus and the number of thymocytes were dramatically reduced compared with those in heterozygous littermates. Further, the compartmentalization into cortex and medulla and the organization of the thymus epithelium were disturbed. The numbers of all thymocyte subpopulations were reduced, with the CD4 and CD8 double-positive thymocytes being most severely affected. The proportion of proliferating thymocytes was reduced, and the staining of apoptotic cells in situ and ex vivo indicated an increased number of apoptotic cells. Isolated thymocytes of Vamp8 (-/-) mice were more susceptible to various apoptotic stimuli including glucocorticoids, FAS receptor, and CD3/CD28-mediated signaling in vitro, even before an increased number of apoptotic cells was detectable in situ. However, bone marrow of phenotypically affected Vamp8 (-/-) mice was readily able to repopulate immunodeficient hosts suggesting that the SNARE protein VAMP8 has a specific function in the thymic stroma affecting the proliferation and apoptosis of T lymphocytes during maturation in the thymus.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , R-SNARE Proteins/genetics , Thymus Gland/abnormalities , Animals , Antibodies, Monoclonal , Apoptosis/drug effects , CD28 Antigens/metabolism , CD3 Complex/metabolism , Dexamethasone/pharmacology , Fas-Associated Death Domain Protein/pharmacology , Glucocorticoids/pharmacology , Hematopoietic Stem Cells/metabolism , Immunologic Factors/pharmacology , Mice , Mice, Knockout , Thymus Gland/immunology , Thymus Gland/ultrastructureABSTRACT
Aminomethyltransferase (Amt), also called glycine cleavage system T-protein is an important enzyme in glycine metabolism (EC 2.1.2.10). Mutations in this gene in humans lead to nonketotic hyperglycinemia, a fatal Mendelian disease. Here, we report the cloning and sequencing of the murine Amt gene. The murine Amt gene consists of nine closely spaced exons that are contained within approximately 5 kb of genomic DNA. It encodes a protein of 403 amino acids that is highly homologous to other mammalian aminomethyltransferases. The cis-acting promoter of the Amt gene is likely to be very short as immediately upstream of the murine Amt gene another gene termed Nicolin 1 gene (Nicn1) is located.
Subject(s)
Hydroxymethyl and Formyl Transferases/genetics , Amino Acid Sequence , Aminomethyltransferase , Animals , Base Composition , Base Sequence , Chromosome Mapping , DNA/analysis , Genomic Library , Hyperglycinemia, Nonketotic/enzymology , Hyperglycinemia, Nonketotic/genetics , Introns , Mice , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
We have identified a novel mammalian gene, termed nicolin 1 gene (NICN1), that is present in human, dog and mouse, whereas it is absent from the available genome sequences of nonmammalian organisms. The NICN1 gene consists of six exons and spans about 6 kb of genomic DNA. It encodes a 213 amino acid protein that does not belong to any known protein family. Experiments using green fluorescent protein (GFP)-tagged nicolin 1 fusion proteins indicate that nicolin 1 is a nuclear protein. Northern analysis and semiquantitative RT-PCR demonstrated that the 2.5 kb NICN1 mRNA is expressed in a tissue-specific manner. The highest NICN1 expression levels are found in brain, testis, liver, and kidney. On the other hand the NICN1 expression is weak in spleen, leukocytes, small intestine and colon. The NICN1 gene is also expressed during development.