RESUMO
The d, ash, and ln coat color mutations provide a unique model system for the study of vesicle transport in mammals. All three mutant loci encode genes that are required for the polarized transport of melanosomes, the specialized, pigment-containing organelles of melanocytes, to the neighboring keratinocytes and eventually into coat hairs. Genetic studies suggest that these genes function in the same or overlapping pathways and are supported by biochemical studies showing that d encodes an actin-based melanosome transport motor, MyoVa, whereas ash encodes Rab27a, a protein that localizes to the melanosome and is postulated to serve as the MyoVa receptor. Here we show that ln encodes melanophilin (Mlph), a previously undescribed protein with homology to Rab effectors such as granuphilin, Slp3-a, and rabphilin-3A. Like all of these effectors, Mlph possesses two Zn(2+)-binding CX(2)CX(13,14)CX(2)C motifs and a short aromatic-rich amino acid region that is critical for Rab binding. However, Mlph does not contain the two Ca(2+)-binding C(2) domains found in these and other proteins involved in vesicle transport, suggesting that it represents a previously unrecognized class of Rab effectors. Collectively, our data show that Mlph is a critical component of the melanosome transport machinery and suggest that Mlph might function as part of a transport complex with Rab27a and MyoVa.
Assuntos
Proteínas de Transporte/genética , Melanossomas/metabolismo , Mutação , Transtornos da Pigmentação/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas Adaptadoras de Transdução de Sinal , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais Bacterianos/genética , Primers do DNA/genética , Teste de Complementação Genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Família Multigênica , Homologia de Sequência de Aminoácidos , Proteínas rab27 de Ligação ao GTPRESUMO
The dilute (d), leaden (ln), and ashen (ash) mutations provide a unique model system for studying vesicle transport in mammals. All three mutations produce a lightened coat color because of defects in pigment granule transport. In addition, all three mutations are suppressed by the semidominant dilute-suppressor (dsu), providing genetic evidence that these mutations function in the same or overlapping transport pathways. Previous studies showed that d encodes a major vesicle transport motor, myosin-VA, which is mutated in Griscelli syndrome patients. Here, using positional cloning and bacterial artificial chromosome rescue, we show that ash encodes Rab27a. Rab GTPases represent the largest branch of the p21 Ras superfamily and are recognized as key players in vesicular transport and organelle dynamics in eukaryotic cells. We also show that ash mice have platelet defects resulting in increased bleeding times and a reduction in the number of platelet dense granules. These defects have not been reported for d and ln mice. Collectively, our studies identify Rab27a as a critical gene for organelle-specific protein trafficking in melanocytes and platelets and suggest that Rab27a functions in both MyoVa dependent and independent pathways.
Assuntos
Cor de Cabelo/genética , Membranas Intracelulares/metabolismo , Melanócitos/metabolismo , Cadeias Pesadas de Miosina , Miosina Tipo V , Proteínas rab de Ligação ao GTP/genética , Albinismo Oculocutâneo , Animais , Transporte Biológico/genética , Plaquetas/patologia , Mapeamento Cromossômico , Grânulos Citoplasmáticos/parasitologia , Modelos Animais de Doenças , Biblioteca Gênica , Teste de Complementação Genética , Proteínas de Filamentos Intermediários/metabolismo , Melanócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Muridae , Ligação Proteica , Splicing de RNA , Pele/citologia , Síndrome , Proteínas rab27 de Ligação ao GTPRESUMO
Non-agouti-lethal 18H (a18H) mice are dark agouti with black pinna hairs. What makes these mice unique is that they develop a spectrum of immunological diseases not seen in other agouti mutant mice. On the JU/Ct background, a18H mice develop an inflammatory disease of the large intestine. On the C57BL/6J background, they develop a fatal disease characterized by pulmonary chronic interstitial inflammation and alveolar proteinosis, inflammation of the glandular stomach and skin resulting in scarring due to constant itching, and hyperplasia of lymphoid cells, haematopoietic cells and the forestomach epithelium. Previous studies suggested that the a18H mutation results from a paracentric inversion that affects two loci: agouti and another, as yet unidentified locus designated itchy (the provisional gene symbol is Itch), that is responsible for the immunological phenotype of a18H mice. Here we confirm that a18H results from an inversion and show that Itch encodes a novel E3 ubiquitin protein ligase, a protein involved in ubiquitin-mediated protein degradation. Our results indicate that ubiquitin-dependent proteolysis is an important mediator of the immune response in vivo and provide evidence for Itch's role in inflammation and the regulation of epithelial and haematopoietic cell growth.
Assuntos
Inversão Cromossômica , Inflamação/genética , Ligases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA , Humanos , Inflamação/enzimologia , Ligases/biossíntese , Ligases/química , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Ubiquitina-Proteína LigasesRESUMO
Mutations at the mouse tottering (tg) locus cause a delayed-onset, recessive neurological disorder resulting in ataxia, motor seizures, and behavioral absence seizures resembling petit mal epilepsy in humans. A more severe allele, leaner (tg(la)), also shows a slow, selective degeneration of cerebellar neurons. By positional cloning, we have identified an alpha1A voltage-sensitive calcium channel gene that is mutated in tg and tg(la) mice. The alpha1A gene is widely expressed in the central nervous system with prominent, uniform expression in the cerebellum. alpha1A expression does not mirror the localized pattern of cerebellar degeneration observed in tg(la) mice, providing evidence for regional differences in biological function of alpha1A channels. These studies define the first mutations in a mammalian central nervous system-specific voltage-sensitive calcium channel and identify the first gene involved in absence epilepsy.
Assuntos
Canais de Cálcio/genética , Epilepsia Tipo Ausência/genética , Ativação do Canal Iônico/genética , Proteínas do Tecido Nervoso/genética , Sequência de Aminoácidos , Animais , Apoptose , Canais de Cálcio/classificação , Canais de Cálcio Tipo N , Canais de Cálcio Tipo P , Canais de Cálcio Tipo Q , Cerebelo/patologia , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cruzamentos Genéticos , Epilepsia Tipo Ausência/etiologia , Hibridização In Situ , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/classificação , Reação em Cadeia da Polimerase , Conformação Proteica , Análise de Sequência de DNARESUMO
Mutations at the Steel (Sl) locus produce pleiotropic effects on viability as well as hematopoiesis, pigmentation and fertility. Several homozygous viable Sl alleles have previously been shown to contain either structural alterations in mast cell growth factor (Mgf) or regulatory mutations that affect expression of the Mgf gene. More severe Sl alleles cause lethality to homozygous embryos and all lethal Sl alleles examined to date contain deletions that remove the entire Mgf coding region. As the timing of the lethality varies from early to late in gestation, it is possible that some deletions may affect other closely linked genes in addition to Mgf. We have analyzed the extent of deleted sequences in seven homozygous lethal Sl alleles. The results of this analysis suggest that late gestation lethality represents the Sl null phenotype and that peri-implantation lethality results from the deletion of at least one essential gene that maps proximal to Sl. We have also examined gene dosage effects of Sl by comparing the phenotypes of mice homozygous and hemizygous for each of four viable Sl alleles. Lastly, we show that certain combinations of the viable Sl alleles exhibit interallelic complementation. Possible mechanisms by which such complementation could occur are discussed.