RESUMO
Uridine 5'-diphosphate (UDP)-glucose is transported into the lumen of the endoplasmic reticulum (ER), and the Arabidopsis nucleotide sugar transporter AtUTr1 has been proposed to play a role in this process; however, different lines of evidence suggest that another transporter(s) may also be involved. Here we show that AtUTr3 is involved in the transport of UDP-glucose and is located at the ER but also at the Golgi. Insertional mutants in AtUTr3 showed no obvious phenotype. Biochemical analysis in both AtUTr1 and AtUTr3 mutants indicates that uptake of UDP-glucose into the ER is mostly driven by these two transporters. Interestingly, the expression of AtUTr3 is induced by stimuli that trigger the unfolded protein response (UPR), a phenomenon also observed for AtUTr1, suggesting that both AtUTr1 and AtUTr3 are involved in supplying UDP-glucose into the ER lumen when misfolded proteins are accumulated. Disruption of both AtUTr1 and AtUTr3 causes lethality. Genetic analysis showed that the atutr1 atutr3 combination was not transmitted by pollen and was poorly transmitted by the ovules. Cell biology analysis indicates that knocking out both genes leads to abnormalities in both male and female germ line development. These results show that the nucleotide sugar transporters AtUTr1 and AtUTr3 are required for the incorporation of UDP-glucose into the ER, are essential for pollen development and are needed for embryo sac progress in Arabidopsis thaliana.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Glucose/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Transporte de Nucleotídeos/metabolismo , Pólen/metabolismo , Difosfato de Uridina/metabolismo , Sequência de Aminoácidos , Arabidopsis/química , Arabidopsis/embriologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Transporte Biológico , Genótipo , Complexo de Golgi/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutação , Proteínas de Transporte de Nucleotídeos/química , Proteínas de Transporte de Nucleotídeos/genética , Pólen/embriologia , Pólen/ultraestrutura , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Low-density lipoprotein receptor-related protein 1 (LRP1) is an endocytic recycling receptor with two cytoplasmic tyrosine-based basolateral sorting signals. Here we show that during biosynthetic trafficking LRP1 uses AP1B adaptor complex to move from a post-TGN recycling endosome (RE) to the basolateral membrane. Then it recycles basolaterally from the basolateral sorting endosome (BSE) involving recognition by sorting nexin 17 (SNX17). In the biosynthetic pathway, Y(29) but not N(26) from a proximal NPXY directs LRP1 basolateral sorting from the TGN. A N(26)A mutant revealed that this NPXY motif recognized by SNX17 is required for the receptor's exit from BSE. An endocytic Y(63)ATL(66) motif also functions in basolateral recycling, in concert with an additional endocytic motif (LL(86,87)), by preventing LRP1 entry into the transcytotic apical pathway. All this sorting information operates similarly in hippocampal neurons to mediate LRP1 somatodendritic distribution regardless of the absence of AP1B in neurons. LRP1 basolateral distribution results then from spatially and temporally segregation steps mediated by recognition of distinct tyrosine-based motifs. We also demonstrate a novel function of SNX17 in basolateral/somatodendritic recycling from a different compartment than AP1B endosomes.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Membrana Celular/metabolismo , Polaridade Celular , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Sinais Direcionadores de Proteínas , Proteínas de Transporte Vesicular/metabolismo , Complexo 1 de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Animais , Linhagem Celular , Endossomos/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Complexo de Golgi/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Dados de Sequência Molecular , Mutação , Neurônios/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/genética , Rede trans-Golgi/metabolismoRESUMO
In non-neuronal cells, inactivation of protein kinase D (PKD) blocks fission of trans-Golgi network (TGN) transport carriers, inducing the appearance of long tubules filled with cargo. We now report on the function of PKD1 in neuronal protein trafficking. In cultured hippocampal pyramidal cells, the transferrin receptor (TfR) and the low-density receptor-related protein (LRP) are predominantly transported to dendrites and excluded from axons. Expression of kinase-inactive PKD1 or its depletion by RNA interference treatment dramatically and selectively alter the intracellular trafficking and membrane delivery of TfR- and LRP-containing vesicles, without inhibiting exit from the TGN or inducing Golgi tubulation. After PKD1 suppression, dendritic membrane proteins are mispackaged into carriers that transport VAMP2; these vesicles are distributed to both axons and dendrites, but are rapidly endocytosed from dendrites and preferentially delivered to the axonal membrane. A kinase-defective mutant of PKD1 lacking the ability to bind diacylglycerol and hence its Golgi localization does not cause missorting of TfR or LRP. These results suggest that in neurons PKD1 regulates TGN-derived sorting of dendritic proteins and hence has a role in neuronal polarity.
Assuntos
Dendritos/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Neurônios/fisiologia , Proteínas Quinases/fisiologia , Receptores da Transferrina/metabolismo , Animais , Células Cultivadas , Dendritos/efeitos dos fármacos , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Hipocampo/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia Confocal/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Quinase C , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Fatores de Tempo , Transfecção/métodos , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Gravação de Videoteipe/métodos , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
Megalin and the low-density lipoprotein (LDL) receptor-related protein (LRP) are two large members of the LDL receptor family that bind and endocytose multiple ligands. The molecular and cellular determinants that dictate the sorting behavior of these receptors in polarized epithelial cells are largely unknown. Megalin is found apically distributed, whereas the limited information on LRP indicates its polarity. We show here that in Madin-Darby canine kidney cells, both endogenous LRP and a minireceptor containing the fourth ligand-binding, transmembrane and LRP cytosolic domains were basolaterally sorted. In contrast, minireceptors that either lacked the cytoplasmic domain or had the tyrosine in the NPTY motif mutated to alanine showed a preferential apical distribution. In LLC-PK1 cells, endogenous megalin was found exclusively in the apical membrane. Studies were also done using chimeric proteins harboring the cytosolic tail of megalin, one with the fourth ligand-binding domain of LRP and the other two containing the green fluorescent protein as the ectodomain and transmembrane domains of either megalin or LRP. Findings from these experiments showed that the cytosolic domain of megalin is sufficient for apical sorting, and that the megalin transmembrane domain promotes association with lipid rafts. In conclusion, we show that LRP and megalin both contain sorting information in their cytosolic domains that directs opposite polarity, basolateral for LRP and apical for megalin. Additionally, we show that the NPTY motif in LRP is important for basolateral sorting and the megalin transmembrane domain directs association with lipid rafts.