RESUMO
Rab guanosine triphosphatases regulate vesicular transport and membrane traffic within eukaryotic cells. Here, a kinesin-like protein that interacts with guanosine triphosphate (GTP)-bound forms of Rab6 was identified. This protein, termed Rabkinesin-6, was localized to the Golgi apparatus and shown to play a role in the dynamics of this organelle. The carboxyl-terminal domain of Rabkinesin-6, which contains the Rab6-interacting domain, inhibited the effects of Rab6-GTP on intracellular transport. Thus, a molecular motor is a potential effector of a Rab protein, and coordinated action between members of these two families of proteins could control membrane dynamics and directional vesicular traffic.
Assuntos
Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Cinesinas/metabolismo , Proteínas rab de Ligação ao GTP , Proteínas ras/metabolismo , Adenosina Trifosfatases/metabolismo , Fosfatase Alcalina/metabolismo , Sequência de Aminoácidos , Transporte Biológico , Retículo Endoplasmático/metabolismo , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Guanosina Trifosfato/metabolismo , Células HeLa , Humanos , Cinesinas/análise , Cinesinas/química , Cinesinas/genética , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Dados de Sequência Molecular , Peso MolecularRESUMO
Several members of the kinesin superfamily are known to play a prominent role in the motor-driven transport processes that occur in mitotic cells. Here we describe a new mitotic human kinesin-like protein, RB6K (Rabkinesin 6), distantly related to MKLP-1. Expression of RB6K is regulated during the cell cycle at both the mRNA and protein level and, similar to cyclin B, shows a maximum during M phase. Isolation of the RB6K promoter allowed identification of a CDE-CHR element and promoter activity was shown to be maximal during M phase. Immunofluorescence microscopy using antibodies raised against RB6K showed a weak signal in interphase Golgi but a 10-fold higher signal in prophase nuclei. During M phase, the newly synthesized RB6K does not colocalise with Rab6. In later stages of mitosis RB6K localized to the spindle midzone and appeared on the midbodies during cytokinesis. The functional significance of this localization during M phase was revealed by antibody microinjection studies which resulted exclusively in binucleate cells, showing a complete failure of cytokinesis. These results substantiate a crucial role for RB6K in late anaphase B and/or cytokinesis, clearly distinct from the role of MKLP-1.
Assuntos
Ciclo Celular/fisiologia , Divisão Celular/fisiologia , Cinesinas/genética , Cinesinas/metabolismo , Regiões 5' não Traduzidas , Sequência de Bases , Ciclo Celular/genética , Divisão Celular/genética , Primers do DNA/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/genética , Mitose/fisiologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regulação para CimaRESUMO
Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.
Assuntos
Processamento Alternativo , Proteínas rab de Ligação ao GTP/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Cinesinas/metabolismo , Dados de Sequência Molecular , Mutação Puntual , Transporte Proteico , Proteínas rab de Ligação ao GTP/metabolismoRESUMO
Rab6 is a small GTP-binding protein that belongs to the Ras superfamily and is involved in intra-Golgi transport. Using a two-hybrid system screen of a mouse brain cDNA library, we have isolated several clones encoding proteins that interact with Rab6. Approximately 60% of the clones identified encoded a new mouse Rab GDP dissociation inhibitor (GDI) isoform. This GDI isoform is distinct from mouse mGDI-1 and mGDI-2, which have been characterized previously, and most likely represents the mouse counterpart of the rat Rab GDI beta isoform. In the two-hybrid system, GDI beta interacts with wild-type Rab6 and Rab5, but not with a GTP-bound Rab6 mutant, or a Rab6 mutant that cannot be post-translationally processed. We further examined whether mouse GDI beta is functional; we show that recombinant mouse GDI beta is able to remove several Rab proteins, including Rab1, Rab2, Rab4, and Rab6, from membranes. The identification of a third GDI isoform in mouse raised the question whether GDI genes belong to a larger multigenic family. We have shown, by Southern blot analysis of genomic DNA, that at least five GDI gene copies exist in both the mouse and rat genomes. In our two-hybrid screen, we have also characterized another clone that specifically interacts with Rab6. This clone was partially sequenced but shows no homology to known sequences. Finally, a third clone, interacting with both Rab5 and Rab6, also appears to encode a novel protein.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas rab de Ligação ao GTP , Proteínas ras/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Clonagem Molecular , Primers do DNA , DNA Complementar , Camundongos , Dados de Sequência Molecular , Ratos , Homologia de Sequência de Aminoácidos , Proteínas ras/química , Proteínas ras/genéticaRESUMO
The Rab6 GTPase regulates intracellular transport at the level of the Golgi apparatus, probably in a retrograde direction. Here, we report the identification and characterization of a novel human Rab6-interacting protein named human GAPCenA (for 'GAP and centrosome-associated'). Primary sequence analysis indicates that GAPCenA displays similarities, within a central 200 amino acids domain, to both the yeast Rab GTPase activating proteins (GAPs) and to the spindle checkpoint proteins Saccharomyces cerevisiae Bub2p and Schizosaccharomyces pombe Cdc16p. We demonstrate that GAPCenA is indeed a GAP, specifically active in vitro on Rab6 and, to a lesser extent, on Rab4 and Rab2 proteins. Immunofluorescence and cell fractionation experiments showed that GAPCenA is mainly cytosolic but that a minor pool is associated with the centrosome. Moreover, GAPCenA was found to form complexes with cytosolic gamma-tubulin and to play a role in microtubule nucleation. Therefore, GAPCenA may be involved in the coordination of microtubule and Golgi dynamics during the cell cycle.