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1.
Eukaryot Cell ; 8(5): 779-89, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19286982

RESUMO

Amphiphysins are proteins thought to be involved in synaptic vesicle endocytosis. Amphiphysins share a common BAR domain, which can sense and/or bend membranes, and this function is believed to be essential for endocytosis. Saccharomyces cerevisiae cells lacking the amphiphysin ortholog Rvs161 are inviable when starved for glucose. Altering sphingolipid levels in rvs161 cells remediates this defect, but how lipid changes suppress remains to be elucidated. Here, we show that the sugar starvation-induced death of rvs161 cells extends to other fermentable sugar carbon sources, and the loss of sphingolipid metabolism suppresses these defects. In all cases, rvs161 cells respond to the starvation signal, elicit the appropriate transcriptional response, and properly localize the requisite sugar transporter(s). However, Rvs161 is required for transporter endocytosis. rvs161 cells accumulate transporters at the plasma membrane under conditions normally resulting in their endocytosis and degradation. Transporter endocytosis requires the endocytosis (endo) domain of Rvs161. Altering sphingolipid metabolism by deleting the very-long-chain fatty acid elongase SUR4 reinitiates transporter endocytosis in rvs161 and rvs161 endo(-) cells. The sphingolipid-dependent reinitiation of endocytosis requires the ubiquitin-regulating factors Doa1, Doa4, and Rsp5. In the case of Doa1, the phospholipase A(2) family ubiquitin binding motif is dispensable. Moreover, the conserved AAA-ATPase Cdc48 and its accessory proteins Shp1 and Ufd1 are required. Finally, rvs161 cells accumulate monoubiquitin, and this defect is remediated by the loss of SUR4. These results show that defects in sphingolipid metabolism result in the reinitiation of ubiquitin-dependent sugar transporter endocytosis and suggest that this event is necessary for suppressing the nutrient starvation-induced death of rvs161 cells.


Assuntos
Metabolismo dos Carboidratos , Proteínas do Citoesqueleto/metabolismo , Endocitose , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Transporte Biológico , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Regulação Fúngica da Expressão Gênica , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo
2.
Artigo em Inglês | MEDLINE | ID: mdl-18391417

RESUMO

The crystal structure of the SH3 domain of rat endophilin A2 has been determined by the multiwavelength anomalous dispersion method and refined at a resolution of 1.70 A to R and R(free) values of 0.196 and 0.217, respectively. The structure adheres to the canonical SH3-domain fold and is highly similar to those of the corresponding domains of endophilins A1 and A3. An intermolecular packing interaction between two molecules in the lattice exploits features that are commonly observed in SH3-domain ligand recognition, including the insertion of a proline side chain into the ligand-binding groove of the protein and the recognition of a basic residue by a cluster of acidic side chains on the RT loop.


Assuntos
Aciltransferases/química , Domínios de Homologia de src , Animais , Células Cultivadas , Cristalografia por Raios X , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos
3.
J Biol Chem ; 280(6): 4270-8, 2005 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-15561700

RESUMO

Loss of function of either the RVS161 or RVS167 Saccharomyces cerevisiae amphiphysin-like gene confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis. We performed a yeast two-hybrid screen using Rvs161p as bait to uncover proteins involved in this sphingolipid-dependent suppressor pathway. In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp, while also elucidating the Rvs167p amino acid domains to which these proteins bind. By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions. Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity. To determine pathway mechanisms further, we examined the localization of Rvs167p in suppressor mutants. These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p. Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M. (1993) Yeast 9, 267-277). Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells. Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton. Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization.


Assuntos
Proteínas do Citoesqueleto/química , Proteínas do Tecido Nervoso/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Esfingolipídeos/metabolismo , Acetiltransferases , Actinas/química , Actinas/metabolismo , Alelos , Western Blotting , Citoesqueleto/metabolismo , Epitopos/química , Deleção de Genes , Glucana Endo-1,3-beta-D-Glucosidase/química , Imunoprecipitação , Óperon Lac , Lipídeos/química , Meiose , Microdomínios da Membrana/química , Proteínas dos Microfilamentos , Microscopia de Fluorescência , Mutação , Fenótipo , Plasmídeos/metabolismo , Prolina/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/química , Frações Subcelulares , Temperatura , Técnicas do Sistema de Duplo-Híbrido , beta-Galactosidase/metabolismo
4.
J Biol Chem ; 277(39): 36152-60, 2002 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-12145310

RESUMO

arv1Delta mutant cells have an altered sterol distribution within cell membranes (Tinkelenberg, A.H., Liu, Y., Alcantara, F., Khan, S., Guo, Z., Bard, M., and Sturley, S. L. (2000) J. Biol. Chem. 275, 40667-40670), and thus it has been suggested that Arv1p may be involved in the trafficking of sterol in the yeast Saccharomyces cerevisiae and also in humans. Here we present data showing that arv1Delta mutants also harbor defects in sphingolipid metabolism. [(3)H]inositol and [(3)H]dihydrosphingosine radiolabeling studies demonstrated that mutant cells had reduced rates of biosynthesis and lower steady-state levels of complex sphingolipids while accumulating certain hydroxylated ceramide species. Phospholipid radiolabeling studies showed that arv1Delta cells harbored defects in the rates of biosynthesis and steady-state levels of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylglycerol. Neutral lipid radiolabeling studies indicated that the rate of biosynthesis and steady-state levels of sterol ester were increased in arv1Delta cells. Moreover, these same studies demonstrated that arv1Delta cells had decreased rates of biosynthesis and steady-state levels of total fatty acid and fatty acid alcohols. Gas chromatography/mass spectrometry analyses examining different fatty acid species showed that arv1Delta cells had decreased levels of C18:1 fatty acid. Additional gas chromatography/mass spectrometry analyses determining the levels of various molecular sterol species in arv1Delta cells showed that mutant cells accumulated early sterol intermediates. Using fluorescence microscopy we found that GFP-Arv1p localizes to the endoplasmic reticulum and Golgi. Interestingly, the heterologous expression of the human ARV1 cDNA suppressed the sphingolipid metabolic defects of arv1Delta cells. We hypothesize that in eukaryotic cells, Arv1p functions in the sphingolipid metabolic pathway perhaps as a transporter of ceramides between the endoplasmic reticulum and Golgi.


Assuntos
Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esfingolipídeos/metabolismo , Esfingosina/análogos & derivados , Ceramidas/metabolismo , DNA Complementar/metabolismo , Retículo Endoplasmático/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Teste de Complementação Genética , Complexo de Golgi/metabolismo , Proteínas de Fluorescência Verde , Humanos , Inositol/metabolismo , Metabolismo dos Lipídeos , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/química , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Esfingosina/metabolismo , Temperatura , Fatores de Tempo
5.
J Biol Chem ; 277(29): 26177-84, 2002 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-12006573

RESUMO

We had previously isolated the temperature-sensitive erg26-1 mutant and characterized the sterol defects in erg26-1 cells (Baudry, K., Swain, E., Rahier, A., Germann, M., Batta, A., Rondet, S., Mandala, S., Henry, K., Tint, G. S., Edlind, T., Kurtz, M., and Nickels, J. T., Jr. (2001) J. Biol. Chem. 276, 12702-12711). We have now determined the defects in sphingolipid metabolism in erg26-1 cells, examined their effects on cell growth, and initiated studies designed to elucidate how might changes in sterol levels coordinately regulate sphingolipid metabolism in Saccharomyces cerevisiae. Using [(3)H]inositol radiolabeling studies, we found that the biosynthetic rate and steady-state levels of specific hydroxylated forms of inositolphosphorylceramides were decreased in erg26-1 cells when compared with wild type cells. [(3)H]Dihydrosphingosine radiolabeling studies demonstrated that erg26-1 cells had decreased levels of the phytosphingosine-derived ceramides that are the direct precursors of the specific hydroxylated inositol phosphorylceramides found to be lower in these cells. Gene dosage experiments using the sphingolipid long chain sphingoid base (LCB) hydroxylase gene, SUR2, suggest that erg26-1 cells may accumulate LCB, thus placing one point of sterol regulation of sphingolipid synthesis possibly at the level of ceramide metabolism. The results from additional genetic studies using the sphingolipid hydroxylase and copper transporter genes, SCS7 and CCC2, respectively, suggest a second possible point of sterol regulation at the level of complex sphingolipid hydroxylation. In addition, [(3)H]inositol radiolabeling of sterol biosynthesis inhibitor-treated wild type cells and late sterol pathway mutants showed that additional blocks in sterol biosynthesis have profound effects on sphingolipid metabolism, particularly sphingolipid hydroxylation state. Finally, our genetic studies in erg26-1 cells using the LCB phosphate phosphatase gene, LBP1, suggest that increasing the levels of the LCB sphingoid base phosphate can remediate the temperature-sensitive phenotype of erg26-1 cells.


Assuntos
Carboxiliases/fisiologia , Colesterol , Saccharomyces cerevisiae/enzimologia , Esfingolipídeos/metabolismo , Carboxiliases/genética , Colestadienóis/metabolismo , Modelos Químicos , Esteróis/metabolismo
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