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1.
Cold Spring Harb Perspect Biol ; 6(5): a016725, 2014 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-24789820

RESUMO

Clathrin is a molecular scaffold for vesicular uptake of cargo at the plasma membrane, where its assembly into cage-like lattices underlies the clathrin-coated pits of classical endocytosis. This review describes the structures of clathrin, major cargo adaptors, and other proteins that participate in forming a clathrin-coated pit, loading its contents, pinching off the membrane as a lattice-enclosed vesicle, and recycling the components. It integrates as much of the structural information as possible at the time of writing into a sketch of the principal steps in coated-pit and coated-vesicle formation.


Assuntos
Membrana Celular/metabolismo , Clatrina/fisiologia , Actinas/fisiologia , Animais , Auxilinas/química , Auxilinas/fisiologia , Transporte Biológico , Clatrina/química , Invaginações Revestidas da Membrana Celular/fisiologia , Dinaminas/química , Dinaminas/fisiologia , Humanos
2.
Development ; 138(6): 1111-20, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21343365

RESUMO

Clathrin has previously been implicated in Drosophila male fertility and spermatid individualization. To understand further the role of membrane transport in this process, we analyzed the phenotypes of mutations in Drosophila auxilin (aux), a regulator of clathrin function, in spermatogenesis. Like partial loss-of-function Clathrin heavy chain (Chc) mutants, aux mutant males are sterile and produce no mature sperm. The reproductive defects of aux males were rescued by male germ cell-specific expression of aux, indicating that auxilin function is required autonomously in the germ cells. Furthermore, this rescue depends on both the clathrin-binding and J domains, suggesting that the ability of Aux to bind clathrin and the Hsc70 ATPase is essential for sperm formation. aux mutant spermatids show a deficit in formation of the plasma membrane during elongation, which probably disrupts the subsequent coordinated migration of investment cones during individualization. In wild-type germ cells, GFP-tagged clathrin localized to clusters of vesicular structures near the Golgi. These structures also contained the Golgi-associated clathrin adaptor AP-1, suggesting that they were Golgi-derived. By contrast, in aux mutant cells, clathrin localized to abnormal patches surrounding the Golgi and its colocalization with AP-1 was disrupted. Based on these results, we propose that Golgi-derived clathrin-positive vesicles are normally required for sustaining the plasma membrane increase necessary for spermatid differentiation. Our data suggest that Aux participates in forming these Golgi-derived clathrin-positive vesicles and that Aux, therefore, has a role in the secretory pathway.


Assuntos
Auxilinas/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Drosophila/fisiologia , Complexo de Golgi/fisiologia , Espermatogênese/fisiologia , Animais , Animais Geneticamente Modificados , Auxilinas/genética , Auxilinas/metabolismo , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Citocinese/genética , Citocinese/fisiologia , Drosophila/embriologia , Drosophila/genética , Drosophila/metabolismo , Embrião não Mamífero , Feminino , Fertilidade/genética , Fertilidade/fisiologia , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Masculino , Modelos Biológicos , Via Secretória/genética , Via Secretória/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Espermatogênese/genética
3.
Proc Natl Acad Sci U S A ; 107(9): 4412-7, 2010 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-20160091

RESUMO

Neuronally expressed auxilin and ubiquitously expressed cyclin-G-dependent kinase (GAK) are homologous proteins that act as cochaperones to support the Hsc70-dependent clathrin uncoating of clathrin-coated vesicles. GAK was previously shown to be essential in mouse during embryonic development and in the adult. We have now engineered an auxilin knockout mouse. Mutant mice had a high rate of early postnatal mortality and surviving pups generally had a lower body weight than wild-type pups, although they had a normal life span. GAK was up-regulated as much as 3-fold in the brains of both surviving neonates and adult mutant mice. An increased number of clathrin-coated vesicles and empty cages were present at knockout synapses both in situ and in primary neuronal cultures. Additionally, clathrin-mediated endocytosis of synaptic vesicles in knockout hippocampal neurons was impaired, most likely due to sequestration of coat components in assembled coats and cages. Collectively, our results demonstrate the specialized role of auxilin in the recycling of synaptic vesicles at synapses, but also show that its function can be partially compensated for by up-regulation of GAK.


Assuntos
Auxilinas/fisiologia , Clatrina/metabolismo , Endocitose , Sinapses/metabolismo , Animais , Auxilinas/genética , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terminações Nervosas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Regulação para Cima
4.
Traffic ; 9(8): 1354-71, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18489706

RESUMO

Auxilin is a cofactor for Hsc70-mediated uncoating of clathrin-coated vesicles (CCVs). However, small interfering RNA (siRNA) knockdown of the ubiquitous auxilin 2 in HeLa cells only moderately impairs clathrin-dependent trafficking. In this study, we show that HeLa cells also express auxilin 1, previously thought to be neuron specific, and that both auxilins need to be depleted for inhibition of clathrin-mediated endocytosis and intracellular sorting. Depleting both auxilins cause an approximately 50% reduction in the number of clathrin-coated pits at the plasma membrane but enhances the association of clathrin and adaptors with intracellular membranes. CCV fractions isolated from auxilin-depleted cells have an approximately 1.5-fold increase in clathrin content and more than fivefold increase in the amount of AP-2 adaptor complex and other endocytic machinery, with no concomitant increase in cargo. In addition, the structures isolated from auxilin-depleted cells are on average smaller than CCVs from control cells and are largely devoid of membrane, indicating that they are not CCVs but membraneless clathrin cages. Similar structures are observed by electron microscopy in intact auxilin-depleted HeLa cells. Together, these findings indicate that the two auxilins have overlapping functions and that they not only facilitate the uncoating of CCVs but also prevent the formation of nonproductive clathrin cages in the cytosol.


Assuntos
Auxilinas/fisiologia , Membrana Celular/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/química , Clatrina/metabolismo , Auxilinas/genética , Citosol/metabolismo , Endocitose , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Choque Térmico HSC70/química , Células HeLa , Humanos , Modelos Biológicos , Neurônios/metabolismo , Transporte Proteico , Interferência de RNA , RNA Interferente Pequeno/metabolismo
5.
Mol Biol Cell ; 19(7): 2766-76, 2008 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-18434600

RESUMO

Hsc70 with its cochaperone, either auxilin or GAK, not only uncoats clathrin-coated vesicles but also acts as a chaperone during clathrin-mediated endocytosis. However, because synaptojanin is also involved in uncoating, it is not clear whether GAK is an essential gene. To answer this question, GAK conditional knockout mice were generated and then mated to mice expressing Cre recombinase under the control of the nestin, albumin, or keratin-14 promoters, all of which turn on during embryonic development. Deletion of GAK from brain, liver, or skin dramatically altered the histology of these tissues, causing the mice to die shortly after birth. Furthermore, by expressing a tamoxifen-inducible promoter to express Cre recombinase we showed that deletion of GAK caused lethality in adult mice. Mouse embryonic fibroblasts in which the GAK was disrupted showed a lack of clathrin-coated pits and a complete block in clathrin-mediated endocytosis. We conclude that GAK deletion blocks development and causes lethality in adult animals by disrupting clathrin-mediated endocytosis.


Assuntos
Auxilinas/fisiologia , Ciclinas/química , Regulação da Expressão Gênica no Desenvolvimento , Proteínas Serina-Treonina Quinases/fisiologia , Animais , Auxilinas/metabolismo , Clatrina/química , Ciclina G , Ciclina G1 , Células-Tronco Embrionárias/citologia , Endocitose , Feminino , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Proteínas Serina-Treonina Quinases/genética , Distribuição Tecidual
6.
Mol Cell ; 28(3): 422-33, 2007 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-17996706

RESUMO

The many protein processing reactions of the ATP-hydrolyzing Hsp70s are regulated by J cochaperones, which contain J domains that stimulate Hsp70 ATPase activity and accessory domains that present protein substrates to Hsp70s. We report the structure of a J domain complexed with a J responsive portion of a mammalian Hsp70. The J domain activates ATPase activity by directing the linker that connects the Hsp70 nucleotide binding domain (NBD) and substrate binding domain (SBD) toward a hydrophobic patch on the NBD surface. Binding of the J domain to Hsp70 displaces the SBD from the NBD, which may allow the SBD flexibility to capture diverse substrates. Unlike prokaryotic Hsp70, the SBD and NBD of the mammalian chaperone interact in the ADP state. Thus, although both nucleotides and J cochaperones modulate Hsp70 NBD:linker and NBD:SBD interactions, the intrinsic persistence of those interactions differs in different Hsp70s and this may optimize their activities for different cellular roles.


Assuntos
Auxilinas/química , Proteínas de Choque Térmico HSP70/química , Chaperonas Moleculares/química , Difosfato de Adenosina/química , Adenosina Trifosfatases/metabolismo , Animais , Auxilinas/fisiologia , Sítios de Ligação , Bovinos , Cristalografia por Raios X , Proteínas de Choque Térmico HSP70/metabolismo , Cinética , Modelos Moleculares , Chaperonas Moleculares/fisiologia , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína
7.
EMBO J ; 25(18): 4163-74, 2006 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-16946707

RESUMO

During clathrin-mediated endocytosis, the GTPase dynamin promotes formation of clathrin-coated vesicles, but its mode of action is unresolved. We provide evidence that a switch in three functional states of dynamin (dimers, tetramers, rings/spirals) coordinates its GTPase cycle. Dimers exhibit negative cooperativity whereas tetramers exhibit positive cooperativity with respect to GTP. Our study identifies tetramers as the kinetically most stable GTP-bound conformation of dynamin, which is required to promote further assembly into higher order structures such as rings or spirals. In addition, using fluorescence lifetime imaging microscopy, we show that interactions between dynamin and auxilin in cells are GTP-, endocytosis- and tetramer-dependent. Furthermore, we show that the cochaperone activity of auxilin is required for constriction of clathrin-coated pits, the same early step in endocytosis known to be regulated by the lifetime of dynamin:GTP. Together, our findings support the model that the GTP-bound conformation of dynamin tetramers stimulates formation of constricted coated pits at the plasma membrane by regulating the chaperone activity of hsc70/auxilin.


Assuntos
Auxilinas/fisiologia , Dinaminas/química , Dinaminas/fisiologia , Endocitose/fisiologia , Animais , Linhagem Celular , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Invaginações Revestidas da Membrana Celular/ultraestrutura , Cães , Guanosina Trifosfato/metabolismo , Humanos , Rim/metabolismo , Rim/ultraestrutura , Camundongos , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Estrutura Quaternária de Proteína , Ratos
8.
J Cell Biol ; 173(3): 443-52, 2006 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-16682530

RESUMO

We have isolated mutations in the Drosophila melanogaster homologue of auxilin, a J-domain-containing protein known to cooperate with Hsc70 in the disassembly of clathrin coats from clathrin-coated vesicles in vitro. Consistent with this biochemical role, animals with reduced auxilin function exhibit genetic interactions with Hsc70 and clathrin. Interestingly, the auxilin mutations interact specifically with Notch and disrupt several Notch-mediated processes. Genetic evidence places auxilin function in the signal-sending cells, upstream of Notch receptor activation, suggesting that the relevant cargo for this auxilin-mediated endocytosis is the Notch ligand Delta. Indeed, the localization of Delta protein is disrupted in auxilin mutant tissues. Thus, our data suggest that auxilin is an integral component of the Notch signaling pathway, participating in the ubiquitin-dependent endocytosis of Delta. Furthermore, the fact that auxilin is required for Notch signaling suggests that ligand endocytosis in the signal-sending cells needs to proceed past coat disassembly to activate Notch.


Assuntos
Auxilinas/fisiologia , Drosophila melanogaster/fisiologia , Proteínas de Membrana/metabolismo , Receptores Notch/fisiologia , Transdução de Sinais/fisiologia , Animais , Auxilinas/genética , Padronização Corporal/genética , Padronização Corporal/fisiologia , Clatrina/genética , Clatrina/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endocitose/genética , Endocitose/fisiologia , Receptores ErbB/genética , Receptores ErbB/fisiologia , Anormalidades do Olho/genética , Anormalidades do Olho/ultraestrutura , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Proteínas de Choque Térmico HSC70/genética , Proteínas de Choque Térmico HSC70/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana/genética , Microscopia Eletrônica de Varredura , Mutação/genética , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Fenótipo , Células Fotorreceptoras de Invertebrados/embriologia , Células Fotorreceptoras de Invertebrados/metabolismo , RNA Interferente Pequeno/genética , Receptores Notch/genética , Transdução de Sinais/genética , Asas de Animais/embriologia , Asas de Animais/metabolismo , Asas de Animais/ultraestrutura
9.
Biochemistry ; 43(11): 3111-9, 2004 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-15023062

RESUMO

The three-dimensional structure of the C-terminal 20 kDa portion of auxilin, which consists of the clathrin binding region and the C-terminal J-domain, has been determined by NMR. Auxilin is an Hsp40 family protein that catalytically supports the uncoating of clathrin-coated vesicles through recruitment of Hsc70 in an ATP hydrolysis-driven process. This 20 kDa auxilin construct contains the minimal sequential region required to uncoat clathrin-coated vesicles catalytically. The tertiary structure consists of six helices, where the first three are unique to auxilin and believed to be important in the catalytic uncoating of clathrin. The last three helices correspond to the canonical J-domain of Hsp40 proteins. The first helix, helix 1, which contains a conserved FEDLL motif believed to be necessary for clathrin binding, is transient and not packed against the rest of the structure. Helix 1 is joined to helix 2 by a flexible linker. Helix 2 packs loosely against the J-domain surface, whereas helix 3 packs tightly and makes critical contributions to the J-domain core. A long insert loop, also unique to the auxilin J-domain, is seen between helix 4 and helix 5. Comparison with a previously reported structure of auxilin containing only helices 3-6 shows a significant difference in the invariant HPD segment of the J-domain. The region where helix 1 is located corresponds to the expected region of the unstructured G/F-rich domain seen in DnaJ, i.e., the canonical N-terminal J-domain protein. In contrast, the location of helix 1 differs from the substrate binding regions of two other Hsp40 proteins, Escherichia coli Hsc20 and viral large T antigen. The variety of biological functions performed by Hsp40 proteins such as auxilin, as well as the observed differences in the structure and function of their substrate binding regions, supports the notion that Hsp40 proteins act as target-specific adaptors that recruit their more general Hsp70 partners to specific biological roles.


Assuntos
Auxilinas/química , Vesículas Revestidas por Clatrina/metabolismo , Fragmentos de Peptídeos/química , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Auxilinas/fisiologia , Catálise , Bovinos , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/enzimologia , Cristalografia por Raios X , Proteínas de Choque Térmico HSC70 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico HSP70/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/fisiologia , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Homologia de Sequência de Aminoácidos , Especificidade por Substrato
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