RESUMO
kinesin-II motor proteins are composed of two different kinesin-like motor proteins and one cargo binding subunit. Here we report the cloning of a new member of the kinesin-II superfamily, Xklp3A from Xenopus laevis, which forms a heterodimeric complex with Xklp3B. The heterodimer formation properties between Xklp3A and B have been tested in vitro using reticulocyte lysate expression and immunoprecipitation. To this end we produced a series of Xklp3A and B constructs of varying length and tested their propensity for heterodimer formation. We could demonstrate that, in contrast to conventional kinesin, the critical domains for heterodimer formation in Xklp3A/B are located at the C-terminal end of the stalk. Neither the neck nor the highly charged stretches after the neck region, which are typical of kinesins-II, are required for heterodimer formation, nor do they prevent homodimer formation. Dimerization is controlled by a cooperative mechanism between the C-terminal coiled-coil segments. Classical trigger sites were not identified. The critical regions for dimerization exhibit a very high degree of sequence conservation among equivalent members of the kinesin-II family.
Assuntos
Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Cinesinas/química , Cinesinas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao Cálcio/genética , Clonagem Molecular , Sequência Conservada , Dimerização , Cinesinas/genética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Musculares/genética , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sequências Repetitivas de Aminoácidos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Proteínas de Xenopus , Xenopus laevisAssuntos
Proteínas de Ligação ao Cálcio/genética , Genes Dominantes , Proteínas Motores Moleculares/genética , Proteínas Musculares/genética , Animais , Biopolímeros/química , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/fisiologia , Células Cultivadas , Clonagem Molecular/métodos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Fibroblastos/química , Fibroblastos/citologia , Corantes Fluorescentes/análise , Complexo de Golgi/metabolismo , Complexo de Golgi/ultraestrutura , Proteínas de Fluorescência Verde , Hemaglutininas Virais/genética , Cinesinas/química , Cinesinas/genética , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Substâncias Macromoleculares , Melaninas/metabolismo , Melanóforos/fisiologia , Microinjeções , Microscopia de Vídeo/métodos , Proteínas Motores Moleculares/fisiologia , Proteínas Musculares/química , Proteínas Musculares/fisiologia , Mutagênese , Reação em Cadeia da Polimerase , Subunidades Proteicas , Relação Estrutura-Atividade , Transfecção , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus , Xenopus laevis , alfa-MSH/farmacologiaRESUMO
The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.
Assuntos
Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Fosfoproteínas , Fuso Acromático/metabolismo , Proteínas de Xenopus , Proteína ran de Ligação ao GTP/metabolismo , Animais , Cromatina/metabolismo , Clonagem Molecular , Feminino , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/isolamento & purificação , GTP Fosfo-Hidrolases/metabolismo , Expressão Gênica/fisiologia , Células HeLa , Humanos , Carioferinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/isolamento & purificação , Microtúbulos/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/isolamento & purificaçãoRESUMO
Melanophores move pigment organelles (melanosomes) from the cell center to the periphery and vice-versa. These bidirectional movements require cytoplasmic microtubules and microfilaments and depend on the function of microtubule motors and a myosin. Earlier we found that melanosomes purified from Xenopus melanophores contain the plus end microtubule motor kinesin II, indicating that it may be involved in dispersion (Rogers, S.L., I.S. Tint, P.C. Fanapour, and V.I. Gelfand. 1997. Proc. Natl. Acad. Sci. USA. 94: 3720-3725). Here, we generated a dominant-negative construct encoding green fluorescent protein fused to the stalk-tail region of Xenopus kinesin-like protein 3 (Xklp3), the 95-kD motor subunit of Xenopus kinesin II, and introduced it into melanophores. Overexpression of the fusion protein inhibited pigment dispersion but had no effect on aggregation. To control for the specificity of this effect, we studied the kinesin-dependent movement of lysosomes. Neither dispersion of lysosomes in acidic conditions nor their clustering under alkaline conditions was affected by the mutant Xklp3. Furthermore, microinjection of melanophores with SUK4, a function-blocking kinesin antibody, inhibited dispersion of lysosomes but had no effect on melanosome transport. We conclude that melanosome dispersion is powered by kinesin II and not by conventional kinesin. This paper demonstrates that kinesin II moves membrane-bound organelles.
Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Cinesinas/fisiologia , Melanóforos/fisiologia , Melanossomas/fisiologia , Proteínas Musculares/fisiologia , Pigmentos Biológicos/metabolismo , Animais , Anticorpos/farmacologia , Proteínas de Ligação ao Cálcio/química , Agregação Celular , Eletroporação , Proteínas de Fluorescência Verde , Cinesinas/química , Proteínas Luminescentes/metabolismo , Lisossomos/fisiologia , Lisossomos/ultraestrutura , Substâncias Macromoleculares , Melanóforos/ultraestrutura , Melanossomas/ultraestrutura , Microscopia de Vídeo , Movimento , Proteínas Musculares/química , Pinocitose , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Xenopus , Proteínas de XenopusRESUMO
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.
