RESUMEN
Mutations in the human dynamin-related protein Drp1 cause mitochondria to form perinuclear clusters. We show here that these mitochondrial clusters consist of highly interconnected mitochondrial tubules. The increased connectivity between mitochondria indicates that the balance between mitochondrial division and fusion is shifted toward fusion. Such a shift is consistent with a block in mitochondrial division. Immunofluorescence and subcellular fractionation show that endogenous Drp1 is localized to mitochondria, which is also consistent with a role in mitochondrial division. A direct role in mitochondrial division is suggested by time-lapse photography of transfected cells, in which green fluorescent protein fused to Drp1 is concentrated in spots that mark actual mitochondrial division events. We find that purified human Drp1 can self-assemble into multimeric ring-like structures with dimensions similar to those of dynamin multimers. The structural and functional similarities between dynamin and Drp1 suggest that Drp1 wraps around the constriction points of dividing mitochondria, analogous to dynamin collars at the necks of budding vesicles. We conclude that Drp1 contributes to mitochondrial division in mammalian cells.
Asunto(s)
GTP Fosfohidrolasas , Proteínas Asociadas a Microtúbulos , Mitocondrias/fisiología , Proteínas/metabolismo , Animales , Células COS , Fraccionamiento Celular , Línea Celular , Citosol/metabolismo , Dinaminas , Genes Reporteros , Humanos , Immunoblotting , Microscopía Fluorescente , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Mutación , Fenotipo , Proteínas/química , Proteínas/genética , Proteínas/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Temperatura , TransfecciónRESUMEN
The roles of mitochondria in cell death and in aging have generated much excitement in recent years. At the same time, however, a quiet revolution in our thinking about mitochondrial ultrastructure has begun. This revolution started with the use of vital dyes and of green fluorescent protein fusion proteins, showing that mitochondria are very dynamic structures that constantly move, divide and fuse throughout the life of a cell. More recently, some of the first proteins contributing to these various processes have been discovered. Our view of the internal structures of mitochondria has also changed. Three-dimensional reconstructions obtained with high voltage electron microscopy show that cristae are often connected to the mitochondrial inner membrane by thin tubules. These new insights are brought to bear on the wealth of data collected by conventional electron microscopic analysis.
Asunto(s)
Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Animales , Transporte Biológico , ADN Mitocondrial/genética , Humanos , Membranas Intracelulares/patología , Fusión de Membrana , Mitocondrias/genética , Mitocondrias/patología , Miopatías Mitocondriales/genética , Miopatías Mitocondriales/patología , Partículas Submitocóndricas/metabolismo , Partículas Submitocóndricas/ultraestructuraAsunto(s)
GTP Fosfohidrolasas/química , GTP Fosfohidrolasas/metabolismo , Técnicas del Sistema de Dos Híbridos , Animales , Baculoviridae/genética , Sitios de Unión , Línea Celular , Reactivos de Enlaces Cruzados/metabolismo , Dimerización , Dinaminas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/aislamiento & purificación , Vectores Genéticos/genética , Glutatión/metabolismo , Glutatión Transferasa/genética , Glutatión Transferasa/metabolismo , Mutagénesis Sitio-Dirigida , Ácidos Ftálicos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Sefarosa/metabolismo , Spodoptera , Transformación Genética , Levaduras/genética , Levaduras/metabolismoRESUMEN
We introduce a computational method for identifying subcellular locations of proteins from the phylogenetic distribution of the homologs of organellar proteins. This method is based on the observation that proteins localized to a given organelle by experiments tend to share a characteristic phylogenetic distribution of their homologs-a phylogenetic profile. Therefore any other protein can be localized by its phylogenetic profile. Application of this method to mitochondrial proteins reveals that nucleus-encoded proteins previously known to be destined for mitochondria fall into three groups: prokaryote-derived, eukaryote-derived, and organism-specific (i.e., found only in the organism under study). Prokaryote-derived mitochondrial proteins can be identified effectively by their phylogenetic profiles. In the yeast Saccharomyces cerevisiae, 361 nucleus-encoded mitochondrial proteins can be identified at 50% accuracy with 58% coverage. From these values and the proportion of conserved mitochondrial genes, it can be inferred that approximately 630 genes, or 10% of the nuclear genome, is devoted to mitochondrial function. In the worm Caenorhabditis elegans, we estimate that there are approximately 660 nucleus-encoded mitochondrial genes, or 4% of its genome, with approximately 400 of these genes contributed from the prokaryotic mitochondrial ancestor. The large fraction of organism-specific and eukaryote-derived genes suggests that mitochondria perform specialized roles absent from prokaryotic mitochondrial ancestors. We observe measurably distinct phylogenetic profiles among proteins from different subcellular compartments, allowing the general use of prokaryotic genomes in learning features of eukaryotic proteins.
Asunto(s)
Proteínas de la Membrana/metabolismo , Filogenia , Señales de Clasificación de Proteína/fisiología , Fracciones Subcelulares/metabolismo , Algoritmos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Dynamin is a 100-kDa GTPase that assembles into multimeric spirals at the necks of budding clathrin-coated vesicles. We describe three different intramolecular binding interactions that may account for the process of dynamin self-assembly. The first binding interaction is the dimerization of a 100-amino acid segment in the C-terminal half of dynamin. We call this segment the assembly domain, because it appears to be critical for multimerization. The second binding interaction occurs between the assembly domain and the N-terminal GTPase domain. The strength of this interaction is controlled by the nucleotide-bound state of the GTPase domain, as shown with mutations in GTP binding motifs and in vitro binding experiments. The third binding interaction occurs between the assembly domain and a segment that we call the middle domain. This is the segment between the N-terminal GTPase domain and the pleckstrin homology domain. The three different binding interactions suggest a model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.
Asunto(s)
GTP Fosfohidrolasas/química , Dinaminas , Modelos Moleculares , Unión Proteica , Estructura Terciaria de Proteína , Levaduras/metabolismoRESUMEN
The function of the GTPase dynamin has been discussed for several years. It clearly plays a role in vesicle budding, but, despite recent insights, precisely how it functions in this process is still a matter of debate. In addition, it is now clear that dynamin is a member of a large protein family, present in a variety of cellular locations where members apparently perform a range of functions. This article describes current understanding of the structure and function of the various dynamin family members.
Asunto(s)
GTP Fosfohidrolasas/fisiología , Secuencia de Aminoácidos , Animales , Dinaminas , GTP Fosfohidrolasas/clasificación , GTP Fosfohidrolasas/metabolismo , Humanos , Datos de Secuencia MolecularRESUMEN
Little is known about the mechanism of mitochondrial division. We show here that mitochondria are disrupted by mutations in a C. elegans dynamin-related protein (DRP-1). Mutant DRP-1 causes the mitochondrial matrix to retract into large blebs that are both surrounded and connected by tubules of outer membrane. This indicates that scission of the mitochondrial outer membrane is inhibited, while scission of the inner membrane still occurs. Overexpressed wild-type DRP-1 causes mitochondria to become excessively fragmented, consistent with an active role in mitochondrial scission. DRP-1 fused to GFP is observed in spots on mitochondria where scission eventually occurs. These data indicate that wild-type DRP-1 contributes to the final stages of mitochondrial division by controlling scission of the mitochondrial outer membrane.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Drosophila , Dinaminas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/metabolismo , Neuropéptidos/metabolismo , Animales , Caenorhabditis elegans/efectos de los fármacos , Caenorhabditis elegans/embriología , Proteínas de Caenorhabditis elegans/genética , División Celular , Linaje de la Célula , Secuencia de Consenso , Dinaminas/genética , Femenino , Expresión Génica/genética , Perfilación de la Expresión Génica , Genes Letales/genética , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/ultraestructura , Proteínas de la Membrana/genética , Mitocondrias/efectos de los fármacos , Mitocondrias/ultraestructura , Datos de Secuencia Molecular , Músculos/citología , Mutación/genética , Neuropéptidos/genética , Oogénesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Acetato de Tetradecanoilforbol/farmacología , Proteínas de Transporte VesicularRESUMEN
Caenorhabditis elegans dynamin is expressed at high levels in neurons and at lower levels in other cell types, consistent with the important role that dynamin plays in the recycling of synaptic vesicles. Indirect immunofluorescence showed that dynamin is concentrated along the dorsal and ventral nerve cords and in the synapse-rich nerve ring. Green fluorescent protein (GFP) fused to the N terminus of dynamin is localized to synapse-rich regions. Furthermore, this chimera was detected along the apical membrane of intestinal cells, in spermathecae, and in coelomocytes. Dynamin localization was not affected by disrupting axonal transport of synaptic vesicles in the unc-104 (kinesin) mutant. To investigate the alternative mechanisms that dynamin might use for translocation to the synapse, we systematically tested the localization of different protein domains by fusion to GFP. Localization of each chimera was measured in one specific neuron, the ALM. The GTPase, a middle domain, and the putative coiled coil each contribute to synaptic localization. Surprisingly, the pleckstrin homology domain and the proline-rich domain, which are known to bind to coated-pit constituents, did not contribute to synaptic localization. The GFP-GTPase chimera was most strongly localized, although the GTPase domain has no known interactions with proteins other than with dynamin itself. Our results suggest that different dynamin domains contribute to axonal transport and the sequestration of a pool of dynamin molecules in synaptic cytosol.
Asunto(s)
Caenorhabditis elegans/metabolismo , GTP Fosfohidrolasas/metabolismo , Animales , Caenorhabditis elegans/enzimología , Dinaminas , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Neuronas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fracciones SubcelularesRESUMEN
Mitochondria exist as a dynamic tubular network with projections that move, break, and reseal in response to local environmental changes. We present evidence that a human dynamin-related protein (Drp1) is specifically required to establish this morphology. Drp1 is a GTPase with a domain structure similar to that of other dynamin family members. To identify the function of Drp1, we transiently transfected cells with mutant Drp1. A mutation in the GTPase domain caused profound alterations in mitochondrial morphology. The tubular projections normally present in wild-type cells were retracted into large perinuclear aggregates in cells expressing mutant Drp1. The morphology of other organelles was unaffected by mutant Drp1. There was also no effect of mutant Drp1 on the transport functions of the secretory and endocytic pathways. By EM, the mitochondrial aggregates found in cells that were transfected with mutant Drp1 appear as clusters of tubules rather than a large mass of coalescing membrane. We propose that Drp1 is important for distributing mitochondrial tubules throughout the cell. The function of this new dynamin-related protein in organelle morphology represents a novel role for a member of the dynamin family of proteins.
Asunto(s)
Proteínas Fúngicas/metabolismo , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/fisiología , Microtúbulos/fisiología , Mitocondrias/enzimología , Proteínas de Saccharomyces cerevisiae , Animales , Transporte Biológico/fisiología , Células COS/química , Células COS/ultraestructura , Clonación Molecular , Gránulos Citoplasmáticos/química , Gránulos Citoplasmáticos/metabolismo , Dinamina I , Dinaminas , Endocitosis/fisiología , Proteínas Fúngicas/genética , GTP Fosfohidrolasas/genética , Regulación Enzimológica de la Expresión Génica , Humanos , Microscopía Electrónica , Mitocondrias/ultraestructura , Proteínas Mitocondriales , Mutagénesis/fisiología , TransfecciónRESUMEN
Drosophila shibire and its mammalian homologue dynamin regulate an early step in endocytosis. We identified a Caenorhabditis elegans dynamin gene, dyn-1, based upon hybridization to the Drosophila gene. The dyn-1 RNA transcripts are trans-spliced to the spliced leader 1 and undergo alternative splicing to code for either an 830- or 838-amino acid protein. These dyn-1 proteins are highly similar in amino acid sequence, structure, and size to the Drosophila and mammalian dynamins: they contain an N-terminal GTPase, a pleckstrin homology domain, and a C-terminal proline-rich domain. We isolated a recessive temperature-sensitive dyn-1 mutant containing an alteration within the GTPase domain that becomes uncoordinated when shifted to high temperature and that recovers when returned to lower temperatures, similar to D. shibire mutants. When maintained at higher temperatures, dyn-1 mutants become constipated, egg-laying defective, and produce progeny that die during embryogenesis. Using a dyn-1::lacZ gene fusion, a high level of dynamin expression was observed in motor neurons, intestine, and pharyngeal muscle. Our results suggest that dyn-1 function is required during development and for normal locomotion.
Asunto(s)
Caenorhabditis elegans/genética , Proteínas de Drosophila , GTP Fosfohidrolasas/fisiología , Mutación de Línea Germinal , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , ADN de Helmintos , Dinaminas , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/metabolismo , Mucosa Intestinal/metabolismo , Datos de Secuencia Molecular , Músculo Liso/metabolismo , Neuronas/metabolismo , Faringe/metabolismo , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
The dynamins are recently discovered GTP-binding proteins postulated to mediate the scission of clathrin-coated vesicles at the plasma membrane. Of the three known mammalian dynamins, dynamin-1 (DNM1) appears to be particularly important for the formation of synaptic vesicles at presynaptic nerve termini. To investigate the possibility that mutations in the DNM1 gene cause a human disease, we determined the chromosomal localization of human DNM1. We conclude from fluorescence in situ hybridization and from the analysis of somatic cell hybrids that the map position in 9q34. This region has syntenic homology with mouse chromosome 2p, in agreement with the map position of the mouse DNM1 gene [see accompanying article by Klocke et al. (1997, Genomics 41:290-292)]. We discuss the potential relevance of the human DNM1 localization to diseases that were mapped genetically to the same chromosomal region.
Asunto(s)
Cromosomas Humanos Par 9 , GTP Fosfohidrolasas/genética , Proteínas de Unión al GTP/genética , Hibridación Fluorescente in Situ , Animales , Células CHO , Cricetinae , Dinamina I , Dinaminas , Humanos , Células HíbridasRESUMEN
A stable HeLa cell line expressing a dynamin mutant, dynts, exhibits a temperature-sensitive defect in endocytic clathrin-coated vesicle formation. Dynts carries a point mutation, G273D, corresponding to the Drosophila shibirets1 allele. The ts-defect in receptor-mediated endocytosis shows a rapid onset (< 5 min) and is readily reversible. At the nonpermissive temperature (38 degrees C) HRP uptake is only partially inhibited. Moreover, when cells are held at the nonpermissive temperature, fluid phase uptake fully recovers to wild-type levels within 30 min, while receptor-mediated endocytosis remains inhibited. The residual HRP uptake early after shift to the nonpermissive temperature and the induced HRP uptake that occurs after recovery are insensitive to cytosol acidification under conditions that potently inhibit receptor-mediated endocytosis of Tfn. Together, these results suggest that a dynamin- and clathrin-independent mechanism contributes to the total constitutive pinocytosis in HeLa cells and that dynts cells rapidly and completely compensate for the loss of clathrin-dependent endocytosis by inducing an alternate endocytic pathway.
Asunto(s)
Clatrina/fisiología , GTP Fosfohidrolasas/genética , Microtúbulos/genética , Pinocitosis/fisiología , Mutación Puntual/fisiología , Invaginaciones Cubiertas de la Membrana Celular/fisiología , Dinaminas , Endocitosis/fisiología , Células HeLa/citología , Células HeLa/fisiología , Humanos , TemperaturaRESUMEN
Src homology 3 (SH3) domains are found in a variety of proteins that are involved in signal transduction or represent components of the cytoskeleton. These domains are thought to serve as modules that mediate specific protein-protein interactions that include proline-rich sequences on the target protein. We have identified proteins of 110, 80, 65, and 43 kDa in human embryonic fibroblasts that bind specifically to the SH3 domain of phospholipase C gamma, a primary substrate of receptor tyrosine kinases, and characterized the 110-kDa band as the microtubule-activated GTPase dynamin. In addition, dynamin binds the son of sevenless adaptor protein GRB-2 with even higher affinity. This interaction does not require the dynamin GTPase function and involves a proline-rich target sequence between residues 812 and 820 of dynamin.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , GTP Fosfohidrolasas/metabolismo , Microtúbulos/enzimología , Proteínas/metabolismo , Fosfolipasas de Tipo C/metabolismo , Secuencia de Aminoácidos , Dinaminas , Proteína Adaptadora GRB2 , Genes src , Glutatión Transferasa/genética , Humanos , Datos de Secuencia Molecular , Unión Proteica , Conformación Proteica , Proteínas Recombinantes de Fusión , Transducción de SeñalRESUMEN
The role of human dynamin in receptor-mediated endocytosis was investigated by transient expression of GTP-binding domain mutants in mammalian cells. Using assays which detect intermediates in coated vesicle formation, the dynamin mutants were found to block endocytosis at a stage after the initiation of coat assembly and preceding the sequestration of ligands into deeply invaginated coated pits. Membrane transport from the ER to the Golgi complex was unaffected indicating that dynamin mutants specifically block early events in endocytosis. These results demonstrate that mutations in the GTP-binding domain of dynamin block Tfn-endocytosis in mammalian cells and suggest that a functional dynamin GTPase is required for receptor-mediated endocytosis via clathrin-coated pits.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/fisiología , Endocitosis , Endosomas/metabolismo , Transferrina/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , ATPasa de Ca(2+) y Mg(2+)/química , ATPasa de Ca(2+) y Mg(2+)/genética , Dinaminas , Retículo Endoplásmico/metabolismo , Endosomas/ultraestructura , Técnica del Anticuerpo Fluorescente , Aparato de Golgi/metabolismo , Células HeLa , Humanos , Datos de Secuencia Molecular , MutaciónRESUMEN
Temperature-sensitive paralysis is the most striking defect of adult Drosophila carrying the shibire mutation. This is believed to be due to a reversible block of endocytosis, which prevents membrane cycling and thus depletes synaptic vesicles. The shibire mutation also affects many tissues outside the nervous system. We have now mapped and characterized the shibire gene. A 275-kilobase yeast artificial chromosome was subcloned into cosmids, among which the gene was then located by analysing with restriction-fragment length polymorphisms. A 15-kilobase fragment of wild-type DNA rescues the mutant phenotype and the sequence of two mutant alleles show differences with wild type, demonstrating that we have isolated the shibire gene. The gene encodes a protein that is highly similar to rat dynamin, 69% of the amino-acid sequence is identical. Dynamin is a GTP-driven mechanochemical enzyme related to mammalian mx-proteins and to the yeast vps 1 gene product. Because the shibire gene product and dynamin have extensive similarity, we propose that they are cognate homologues. Dynamin causes microtubules to slide along each other in vitro and in extracts it is associated with a distinct, but so far uncharacterized, membrane fraction. In light of the shibire phenotype, we suggest that these proteins provide the motor for vesicular transport during endocytosis.
Asunto(s)
ATPasa de Ca(2+) y Mg(2+)/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Secuencia de Aminoácidos , Animales , Membrana Celular/fisiología , Clonación Molecular , Dinaminas , Endocitosis , Genes , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Mapeo RestrictivoRESUMEN
We have transfected a eukaryotic expression vector containing a mdr1 complementary DNA isolated from normal human liver into human BRO melanoma cells to study the drug-resistant phenotype produced by the exclusive overexpression of normal human mdr1 P-glycoprotein. The drug resistance pattern of mdr1-transfected clones includes relatively high resistance to gramicidin D (about 300-fold), vincristine (about 100-fold), and actinomycin D (about 100-fold) and a lower degree of resistance to doxorubicin (about 10-fold), VP16-213 (about 10-fold), and colchicine (about 6-fold). The transfectants did not exhibit resistance to trimetrexate, cis-platinum, mitomycin C, 1-beta-D-arabinofuranosylcytosine, bleomycin, G418, or magainin-2-amide; they were slightly more sensitive to verapamil (2-fold) but not to Triton X-100. As in other multidrug-resistant cell lines, resistance to vincristine could be reversed by verapamil and, more effectively, by cyclosporin A. Chloroquine only marginally increased drug sensitivity in mdr1-transfected cells. Gramicidin D resistance was also reversed by verapamil, suggesting that the mechanism of resistance to this polypeptide antibiotic is similar to that of other drugs transported by P-glycoprotein. Thus, expression of the wild-type mdr1 complementary DNA induces a drug-resistant phenotype similar to that induced by mdr1 complementary DNAs isolated from drug-resistant cell lines with relatively low colchicine resistance. As other cell lines may display a different pattern of drug resistance, it is clear that other resistance mechanisms or cell type-specific factors may modulate the resistance. mdr1-transfected cell lines provide a convenient tool for the identification of P-glycoprotein-mediated phenomena.
Asunto(s)
Antineoplásicos/farmacología , Resistencia a Medicamentos/genética , Glicoproteínas de Membrana/genética , Transfección , Células Tumorales Cultivadas/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Transporte Biológico , División Celular/efectos de los fármacos , Línea Celular , Membrana Celular/metabolismo , ADN/genética , Daunorrubicina/metabolismo , Humanos , Cinética , Melanoma , Hibridación de Ácido Nucleico , Fenotipo , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/metabolismoRESUMEN
We have determined the sequence of the human mdr3 gene using cDNA derived from liver RNA. The mdr3 gene codes for a member of a family of membrane proteins, the P-glycoproteins, overproduced in many multi-drug-resistant (MDR) cell lines. Like its relatives, the protein encoded by mdr3 has a deduced Mr of 140,000, which is presumably increased by glycosylation after synthesis. The sequence consists of two similar halves, each with a series of six hydrophobic segments that may form a membrane channel. The halves also possess nucleotide-binding consensus sequences, which presumably act as ATPases and drive drug transport. The presumed ATPase domains are all but identical to those of the human mdr1 gene product [Chen et al., Cell 47 (1986) 381-389]. We attribute this high level of sequence conservation to the repeated gene conversion that is evident from segments in which mdr1 and mdr3 differ only in a few silent mutations. Divergence between P-glycoprotein family members is greatest at the N terminus and in the 60 amino acid linker connecting the two halves. In the putative trans-membrane domains approx. 80% of the amino acids are conserved between the products of mdr1 and mdr3. Although the function of mdr3 is not yet known, its high homology with mdr1 suggests that it also encodes an efflux pump with broad specificity.