Mitochondrial telomere-binding protein from Candida parapsilosis suggests an evolutionary adaptation of a nonspecific single-stranded DNA-binding protein.
(17/6694)
The mitochondrial genome in a number of organisms is represented by linear DNA molecules with defined terminal structures. The telomeres of linear mitochondrial DNA (mtDNA) of yeast Candida parapsilosis consist of tandem arrays of large repetitive units possessing single-stranded 5' extension of about 110 nucleotides. Recently we identified the first mitochondrial telomere-binding protein (mtTBP) that specifically binds a sequence derived from the extreme end of C. parapsilosis linear mtDNA and protects it from attack by various DNA-modifying enzymes (Tomaska, L'., Nosek, J., and Fukuhara, H. (1997) J. Biol. Chem. 272, 3049-3059). Here we report the isolation of MTP1, the gene encoding mtTBP of C. parapsilosis. Sequence analysis revealed that mtTBP shares homology with several bacterial and mitochondrial single-stranded DNA-binding proteins that nonspecifically bind to single-stranded DNA with high affinity. Recombinant mtTBP displays a preference for the telomeric 5' overhang of C. parapsilosis mtDNA. The heterologous expression of a mtTBP-GFP fusion protein resulted in its localization to the mitochondria but was unable to functionally substitute for the loss of the S. cerevisiae homologue Rimlp. Analysis of the MTP1 gene and its translation product mtTBP may provide an insight into the evolutionary origin of linear mitochondrial genomes and the role it plays in their replication and maintenance. (+info)
Ligand binding properties of the very low density lipoprotein receptor. Absence of the third complement-type repeat encoded by exon 4 is associated with reduced binding of Mr 40,000 receptor-associated protein.
(18/6694)
The very low density lipoprotein receptor (VLDLR) binds, among other ligands, the Mr 40,000 receptor-associated protein (RAP) and a variety of serine proteinase-serpin complexes, including complexes of the proteinase urokinase-type plasminogen activator (uPA) with the serpins plasminogen activator inhibitor-1 (PAI-1) and protease nexin-1 (PN-1). We have analyzed the binding of RAP, uPA.PAI-1, and uPA.PN-1 to two naturally occurring VLDLR variants, VLDLR-I, containing all eight complement-type repeats, and VLDLR-III, lacking the third complement-type repeat, encoded by exon 4. VLDLR-III displayed approximately 4-fold lower binding of RAP than VLDLR-I and approximately 10-fold lower binding of the most C-terminal one of the three domains of RAP. In contrast, the binding of uPA.PAI-1 and uPA.PN-1 to the two VLDLR variants was indistinguishable. Surprisingly, uPA.PN-1, but not uPA.PAI-1, competed RAP binding to both VLDLR variants. These observations show that the third complement-type repeat plays a crucial role in maintaining the contact sites needed for optimal recognition of RAP, but does not affect the proteinase-serpin complex contact sites, and that two ligands can show full cross-competition without sharing the same contacts with the receptor. These results elucidate the mechanisms of molecular recognition of ligands by receptors of the low density lipoprotein receptor family. (+info)
Mutant vasopressin precursors that cause autosomal dominant neurohypophyseal diabetes insipidus retain dimerization and impair the secretion of wild-type proteins.
(19/6694)
Autosomal dominant familial neurohypophyseal diabetes insipidus is caused by mutations in the arginine vasopressin (AVP) gene. We demonstrated recently that mutant AVP precursors accumulate within the endoplasmic reticulum of neuronal cells, leading to cellular toxicity. In this study, the possibility that mutant AVP precursors interact with wild-type (WT) proteins to alter their processing and function was explored. WT and mutant precursors were epitope-tagged to allow them to be distinguished in transfected cells. An in vivo cross-linking reaction revealed homo- and heterodimer formation between WT and mutant precursors. Mutant precursors were also shown to impair intracellular trafficking of WT precursors from the endoplasmic reticulum to the Golgi apparatus. In addition to the cytotoxicity caused by mutant AVP precursors, the interaction between the WT and mutant precursors suggests that a dominant-negative mechanism may also contribute to the pathogenesis of familial neurohypophyseal diabetes insipidus. (+info)
Nucleo-cytoplasmic interactions that control nuclear envelope breakdown and entry into mitosis in the sea urchin zygote.
(20/6694)
In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus. (+info)
Dynamic association of L-selectin with the lymphocyte cytoskeletal matrix.
(21/6694)
L-selectin mediates lymphocyte extravasation into lymphoid tissues through binding to sialomucin-like receptors on the surface of high endothelial venules (HEV). This study examines the biochemical basis and regulation of interactions between L-selectin, an integral transmembrane protein, and the lymphocyte cytoskeleton. Using a detergent-based extraction procedure, constitutive associations between L-selectin and the insoluble cytoskeletal matrix could not be detected. However, engagement of the L-selectin lectin domain by Abs or by glycosylation-dependent cell adhesion molecule-1, an HEV-derived ligand for L-selectin, rapidly triggered redistribution of L-selectin to the detergent-insoluble cytoskeleton. L-selectin attachment to the cytoskeleton was not prevented by inhibitors of actin/microtubule polymerization (cytochalasin B, colchicine, or nocodozole) or serine/threonine and tyrosine kinase activity (staurosporine, calphostin C, or genistein), although L-selectin-mediated adhesion of human PBL was markedly suppressed by these agents. Exposure of human PBL or murine pre-B transfectants expressing full-length human L-selectin to fever-range hyperthermia also markedly increased L-selectin association with the cytoskeleton, directly correlating with enhanced L-selectin-mediated adhesion. In contrast, a deletion mutant of L-selectin lacking the COOH-terminal 11 amino acids failed to associate with the cytoskeletal matrix in response to Ab cross-linking or hyperthermia stimulation and did not support adhesion to HEV. These studies, when taken together with the previously demonstrated interaction between the L-selectin cytoplasmic domain and the cytoskeletal linker protein alpha-actinin, strongly implicate the actin-based cytoskeleton in dynamically controlling L-selectin adhesion. (+info)
Exonic splicing enhancers contribute to the use of both 3' and 5' splice site usage of rat beta-tropomyosin pre-mRNA.
(22/6694)
The rat beta-tropomyosin gene encodes two tissue-specific isoforms that contain the internal, mutually exclusive exons 6 (nonmuscle/smooth muscle) and 7 (skeletal muscle). We previously demonstrated that the 3' splice site of exon 6 can be activated by introducing a 9-nt polyuridine tract at its 3' splice site, or by strengthening the 5' splice site to a U1 consensus binding site, or by joining exon 6 to the downstream common exon 8. Examination of sequences within exons 6 and 8 revealed the presence of two purine-rich motifs in exon 6 and three purine-rich motifs in exon 8 that could potentially represent exonic splicing enhancers (ESEs). In this report we carried out substitution mutagenesis of these elements and show that some of them play a critical role in the splice site usage of exon 6 in vitro and in vivo. Using UV crosslinking, we have identified SF2/ASF as one of the cellular factors that binds to these motifs. Furthermore, we show that substrates that have mutated ESEs are blocked prior to A-complex formation, supporting a role for SF2/ASF binding to the ESEs during the commitment step in splicing. Using pre-mRNA substrates containing exons 5 through 8, we show that the ESEs within exon 6 also play a role in cooperation between the 3' and 5' splice sites flanking this exon. The splicing of exon 6 to 8 (i.e., 5' splice site usage of exon 6) was enhanced with pre-mRNAs containing either the polyuridine tract in the 3' splice site or consensus sequence in the 5' splice site around exon 6. We show that the ESEs in exon 6 are required for this effect. However, the ESEs are not required when both the polyuridine and consensus splice site sequences around exon 6 were present in the same pre-mRNA. These results support and extend the exon-definition hypothesis and demonstrate that sequences at the 3' splice site can facilitate use of a downstream 5' splice site. In addition, the data support the hypothesis that ESEs can compensate for weak splice sites, such as those found in alternatively spliced exons, thereby providing a target for regulation. (+info)
Cross-linking of the B cell receptor induces activation of phospholipase D through Syk, Btk and phospholipase C-gamma2.
(23/6694)
Phospholipase D (PLD) has been proposed to play a key role in the signal transduction of cellular responses to various extracellular signals. Herein we provide biochemical and genetic evidence that cross-linking of the B cell receptor (BCR) induces rapid activation of PLD through a Syk-, Btk- and phospholipase C (PLC)-gamma2-dependent pathway in DT40 cells. Activation of PLD upon BCR engagement is completely blocked in Syk- or Btk-deficient cells, but unaffected in Lyn-deficient cells. Furthermore, in PLC-gamma2-deficient cells, BCR engagement failed to activate PLD. These results demonstrate that Syk, Btk and PLC-gamma2 are essential for BCR-induced PLD activation. (+info)
Thiol oxidation of actin produces dimers that enhance the elasticity of the F-actin network.
(24/6694)
Slow oxidation of sulfhydryls, forming covalently linked actin dimers and higher oligomers, accounts for increases in the shear elasticity of purified actin observed after aging. Disulfide-bonded actin dimers are incorporated into F-actin during polymerization and generate cross-links between actin filaments. The large gel strength of oxidized actin (>100 Pa for 1 mg/ml) in the absence of cross-linking proteins falls to within the theoretically predicted order of magnitude for uncross-linked actin filament networks (1 Pa) with the addition of sufficient concentrations of reducing agents such as 5 mM dithiothreitol or 10 mM beta-mercaptoethanol. As little as 1 gelsolin/1000 actin subunits also lowers the high storage modulus of oxidized actin. The effects of gelsolin may be both to increase filament number as it severs F-actin and to cover the barbed end of an actin filament, which otherwise might cross-link to the side of another filament via an actin dimer. These new findings may explain why previous studies of actin rheology report a wide range of values when purified actin is polymerized without added regulatory proteins. (+info)