Reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of N-acetylglucosamine

Balch, William E.; Dunphy, William G.; Braell, William A.; Rothman, James E.

doi:10.1016/0092-8674(84)90019-9

Cited by 666 publications

(483 citation statements)

References 27 publications

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“…Golgi fractions from knockin cells were prepared at 4°C as described in Balch et al (1984), with few modifications (Fernandez-Ulibarri et al, 2007). Briefly, twenty-two 100-mm plates (NUNC A/S) per condition were pelleted and washed twice in cold PBS (10 min at 500 ϫ g) and lysis buffer (10 mM Tris-HCl, 0.25 M sucrose, 1 mM phenylmethylsulfonyl fluoride [PMSF], 10 g/ml aprotinin, and 1 g/ml leupeptin, pH 7.4).…”

Section: Isolation Of Golgi Membranesmentioning

confidence: 99%

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Toro

Alberch

Lázaro‐Diéguez

et al. 2009

MBoC

149

117

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Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function.

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Section: Isolation Of Golgi Membranesmentioning

confidence: 99%

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Toro

Alberch

Lázaro‐Diéguez

et al. 2009

MBoC

149

117

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“…Golgi membranes were prepared by a sucrose density method reported previously (Balch et al, 1994), with a protease inhibitor cocktail presented in all buffer solution. The band at the interface of 0.8 and 1.2 M sucrose was collected and subjected to 6% SDS PAGE and immunoblotting as described above.…”

Section: Immunoblot Analysis and Preparation Of Crude Golgi Membranementioning

confidence: 99%

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

Song

Ailenberg

Silverman

1999

MBoC

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We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

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“…Similar in vitro assays have been developed for reconstituting transport and fusion processes, such as ER-to-Golgi transport (Beckers et al, 1987;Baker et al, 1988), transport through the Golgi stack (Balch et al, 1984), nuclear envelope assembly (Boman et al, 1992), and endosome fusion (Mayorga et al, 1989a). Most of these assays are either based on: (a) the mixing of luminal contents of such vesicles (measured by glycosylation of reporter proteins); (b) the formarion of immune complexes (quantified after coprecipitation); or (c) the increase in size of fused vesicles (measured microscopically; for a review on these methods see Pryer et al, 1992).…”

mentioning

confidence: 99%

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

Haas

Conradt

Wickner

1994

The Journal of Cell Biology

166

209

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Abstract. During budding in Saccharomyces cerevisiae, maternal vacuole material is delivered into the growing daughter cell via tubular or vesicular structures. One of the late steps in vacuole inheritance is the fusion in the bud of vesicles derived from the maternal vacuole. This process has been reconstituted in vitro and requires isolated vacuoles, a physiological temperature, cytosolic factors, and ATP (Conradt, B., J. Shaw, T. Vida, S. Emr, and W. Wickner. 1992. J.Cell Biol. 119:1469-1479). We now report a simple and reliable assay to quantify vacuole-to-vacuole fusion in vitro. This assay is based on the maturation and activation of vacuole membrane-bound pro-alkaline phosphatase by vacuolar proteinase A after vacuole-tovacuole fusion. In vitro fusion allowed maturation of 30 to 60% of pro-alkaline phosphatase. Vacuoles prepared from a mutant defective in vacuole inheritance in vivo (vac2-1) were inactive in this assay. Vacuole fusion in vitro required a vacuole membrane potential. Inhibition by nonhydrolyzable guanosine derivatives, mastoparans, and benzalkonium chloride suggest that GTP-hydrolyzing G proteins may play a key role in the in vitro fusion events.

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Reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of N-acetylglucosamine

Cited by 666 publications

References 27 publications

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

Human munc13 Is a Diacylglycerol Receptor that Induces Apoptosis and May Contribute to Renal Cell Injury in Hyperglycemia

G-protein ligands inhibit in vitro reactions of vacuole inheritance.

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