Beclin–phosphatidylinositol 3‐kinase complex functions at the
            trans
            ‐Golgi network

Mannose 6-phosphate receptors carry newly synthesized lysosomal hydrolases from the trans-Golgi network to endosomes, then return to the trans-Golgi network for another round of enzyme delivery. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, interferes with the delivery of newly synthesized lysosomal enzymes to lysosomes. We used two independent assays of mannose 6-phosphate receptor trafficking to determine the precise step that is blocked by wortmannin. Using an assay that monitors resialylation of desialylated cell surface 300-kDa mannose 6-phosphate receptors, we found that receptor endocytosis and transport to the trans-Golgi network were not inhibited by 2 microM wortmannin. In addition, this concentration of drug had no effect on the transport of the mannose 6-phosphate receptor from late endosomes to the trans-Golgi network using a system that reconstitutes this transport process in cell extracts. Under the same conditions, wortmannin significantly inhibited the generation of mature cathepsin D. In addition, the structurally unrelated phosphatidylinositol 3-kinase inhibitor, LY294002, was also without effect when added to in vitro endosome-trans-Golgi network transport reactions. These experiments demonstrate that the interruption in lysosomal enzyme targeting is most likely due to a wortmannin-sensitive process required for the export of these receptors from the trans-Golgi network, consistent with the established role of phosphatidylinositol 3-kinase in the equivalent transport process in Saccharomyces cerevisiae.

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Trafficking of Phosphatidylinositol 3-Phosphate from the trans-Golgi Network to the Lumen of the Central Vacuole in Plant Cells

The Plant Cell ◽

10.2307/3871277 ◽

2001 ◽

Vol 13 (2) ◽

pp. 287 ◽

Cited By ~ 1

Author(s):

Dae Heon Kim ◽

Young-Jae Eu ◽

Cheol Min Yoo ◽

Yong-Woo Kim ◽

Kyeong Tae Pih ◽

...

Keyword(s):

Plant Cells ◽

Central Vacuole ◽

Trans Golgi Network ◽

Golgi Network ◽

Phosphatidylinositol 3

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Wortmannin causes mistargeting of procathepsin D. evidence for the involvement of a phosphatidylinositol 3-kinase in vesicular transport to lysosomes.

The Journal of Cell Biology ◽

10.1083/jcb.130.4.797 ◽

1995 ◽

Vol 130 (4) ◽

pp. 797-805 ◽

Cited By ~ 136

Author(s):

H W Davidson

Keyword(s):

Mammalian Cells ◽

Vesicular Transport ◽

Phosphatidylinositol 3 Kinase ◽

Lysosomal Hydrolases ◽

Ligand Complex ◽

Receptor Ligand ◽

Rapid Restoration ◽

K 562 Cells ◽

Golgi Network ◽

Phosphatidylinositol 3

At present little is known of the biochemical machinery controlling transport of newly synthesized lysosomal hydrolases from the trans-Golgi network (TGN) to endosomes. The demonstration that Vps34p (a protein required for targeting soluble hydrolases to the vacuole in Saccharomyces cerevisiae) is a phosphatidylinositol 3-kinase (PI3-K) suggested the possibility that a homologous enzyme might be involved in the equivalent step in mammalian cells. Using the PI3-K inhibitors wortmannin and LY294002, I provide evidence to support this hypothesis. Treatment of K-562 cells with wortmannin induced secretion of procathepsin D, with half-maximal inhibition of accurate targeting to lysosomes at 10-20 nM. Kinetic analysis indicated that a late Golgi (TGN) step was affected, and that other constitutive vesicular transport events were not. The M6P recognition signal was still generated in the presence of wortmannin suggesting that the drug was directly inhibiting export of the receptor-ligand complex from the TGN, while removal of the drug led to a rapid restoration of accurate sorting. At the concentrations used, wortmannin and LY294002 are presently accepted to be specific inhibitors of PI3-K. I conclude that these data implicate such an enzyme in the trafficking of M6P-receptor-ligand complexes from the TGN towards lysosomes.

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Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The Journal of Cell Biology ◽

10.1083/jcb.200804048 ◽

2008 ◽

Vol 183 (3) ◽

pp. 513-526 ◽

Cited By ~ 262

Author(s):

Raul Rojas ◽

Thijs van Vlijmen ◽

Gonzalo A. Mardones ◽

Yogikala Prabhu ◽

Adriana L. Rojas ◽

...

Keyword(s):

Cathepsin D ◽

Retrograde Transport ◽

Phosphatidylinositol 3 Kinase ◽

Sorting Nexin ◽

Retromer Complex ◽

Endosomal Membranes ◽

Guanosine Triphosphatase ◽

Vacuolar Protein ◽

Golgi Network ◽

Phosphatidylinositol 3

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.

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