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

1995 ◽  
Vol 130 (4) ◽  
pp. 797-805 ◽  
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.

scholarly journals Phosphatidylinositol 3-kinase is not required for recycling of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network.

1997 ◽  
Vol 8 (4) ◽  
pp. 577-582 ◽  
Author(s):  
Y Nakajima ◽  
S R Pfeffer
Keyword(s):  
Transport Process ◽  
Kinase Inhibitor ◽  
Phosphatidylinositol 3 Kinase ◽  
Lysosomal Hydrolases ◽  
Trans Golgi Network ◽  
Cell Extracts ◽  
Late Endosomes ◽  
Mannose 6 Phosphate ◽  
Golgi Network ◽  
Phosphatidylinositol 3

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.

Schizosaccharomyces pombe Vps34p, a phosphatidylinositol-specific PI 3-kinase essential for normal cell growth and vacuole morphology

1995 ◽  
Vol 108 (12) ◽  
pp. 3745-3756 ◽  
Author(s):  
K. Takegawa ◽  
D.B. DeWald ◽  
S.D. Emr
Keyword(s):  
Amino Acid ◽  
Schizosaccharomyces Pombe ◽  
Mammalian Cells ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Phosphatidylinositol 3 Kinase ◽  
Acid Protein ◽  
Phosphatidylinositol 3

We have cloned the gene, vps34+, from the fission yeast Schizosaccharomyces pombe which encodes an 801 amino acid protein with phosphatidylinositol 3-kinase activity. The S. pombe Vps34 protein shares 43% amino acid sequence identity with the Saccharomyces cerevisiae Vps34 protein and 28% identity with the p110 catalytic subunit of the mammalian phosphatidylinositol 3-kinase. When the vps34+ gene is disrupted, S.pombe strains are temperature-sensitive for growth and the mutant cells contain enlarged vacuoles. Furthermore, while wild-type strains exhibit substantial levels of phosphatidylinositol 3-kinase activity, this activity is not detected in the vps34 delta strain. S.pombe Vps34p-specific antiserum detects a single protein in cells of -90 kDa that fractionates almost exclusively with the crude membrane fraction. Phosphatidylinositol 3-kinase activity also is localized mainly in the membrane fraction of wild-type cells. Immunoisolated Vps34p specifically phosphorylates phosphatidylinositol on the D-3 position of the inositol ring to yield phosphatidylinositol(3)phosphate. but does not utilize phosphatidylinositol(4)phosphate or phosphatidylinositol(4,5)bisphosphate as substrates. In addition, when compared to the mammalian p110 phosphatidylinositol 3-kinase, S. pombe Vps34p is relatively insensitive to the inhibitors wortmannin and LY294002. Together, these results indicate that S. pombe Vps34 is more similar to the phosphatidylinositol-specific 3-kinase, Vps34p from S. cerevisiae, and is distinct from the p110/p85 and G protein-coupled phosphatidylinositol 3-kinases from mammalian cells. These data are discussed in relation to the possible role of Vps34p in vesicle-mediated protein sorting to the S. pombe vacuole.

scholarly journals Beclin 1 Forms Two Distinct Phosphatidylinositol 3-Kinase Complexes with Mammalian Atg14 and UVRAG

2008 ◽  
Vol 19 (12) ◽  
pp. 5360-5372 ◽  
Author(s):  
Eisuke Itakura ◽  
Chieko Kishi ◽  
Kinji Inoue ◽  
Noboru Mizushima
Keyword(s):  
Membrane Trafficking ◽  
Mammalian Cells ◽  
Coiled Coil ◽  
Pi3 Kinase ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Interacting Protein ◽  
Vacuolar Protein ◽  
Phosphatidylinositol 3

Class III phosphatidylinositol 3-kinase (PI3-kinase) regulates multiple membrane trafficking. In yeast, two distinct PI3-kinase complexes are known: complex I (Vps34, Vps15, Vps30/Atg6, and Atg14) is involved in autophagy, and complex II (Vps34, Vps15, Vps30/Atg6, and Vps38) functions in the vacuolar protein sorting pathway. Atg14 and Vps38 are important in inducing both complexes to exert distinct functions. In mammals, the counterparts of Vps34, Vps15, and Vps30/Atg6 have been identified as Vps34, p150, and Beclin 1, respectively. However, orthologues of Atg14 and Vps38 remain unknown. We identified putative mammalian homologues of Atg14 and Vps38. The Vps38 candidate is identical to UV irradiation resistance-associated gene (UVRAG), which has been reported as a Beclin 1-interacting protein. Although both human Atg14 and UVRAG interact with Beclin 1 and Vps34, Atg14, and UVRAG are not present in the same complex. Although Atg14 is present on autophagic isolation membranes, UVRAG primarily associates with Rab9-positive endosomes. Silencing of human Atg14 in HeLa cells suppresses autophagosome formation. The coiled-coil region of Atg14 required for binding with Vps34 and Beclin 1 is essential for autophagy. These results suggest that mammalian cells have at least two distinct class III PI3-kinase complexes, which may function in different membrane trafficking pathways.

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

EMBO Reports ◽  
2001 ◽  
Vol 2 (4) ◽  
pp. 330-335 ◽  
Author(s):  
Akio Kihara ◽  
Yukiko Kabeya ◽  
Yoshinori Ohsumi ◽  
Tamotsu Yoshimori
Keyword(s):  
Phosphatidylinositol 3 Kinase ◽  
Trans Golgi Network ◽  
Complex Functions ◽  
Golgi Network ◽  
Phosphatidylinositol 3

scholarly journals Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

2015 ◽  
Vol 26 (6) ◽  
pp. 1119-1128 ◽  
Author(s):  
Bjorn D. M. Bean ◽  
Michael Davey ◽  
Jamie Snider ◽  
Matthew Jessulat ◽  
Viktor Deineko ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Guanine Nucleotide Exchange Factors ◽  
Guanine Nucleotide ◽  
Nucleotide Exchange ◽  
Phosphatidylinositol 3 Kinase ◽  
Endosomal Membrane ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factors ◽  
New Yeast ◽  
Phosphatidylinositol 3

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.

scholarly journals The p110αand p110βIsoforms of Class I Phosphatidylinositol 3-Kinase Are Involved in Toll-Like Receptor 5 Signaling in Epithelial Cells

2010 ◽  
Vol 2010 ◽  
pp. 1-12 ◽  
Author(s):  
Sabine M. Ivison ◽  
Mohammed A. S. Khan ◽  
Nicholas R. Graham ◽  
Leila A. Shobab ◽  
Yu Yao ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
P38 Map Kinase ◽  
Carcinoma Cells ◽  
Model Systems ◽  
Toll Like Receptor ◽  
Phosphatidylinositol 3 Kinase ◽  
Proinflammatory Response ◽  
Colon Carcinoma Cells ◽  
Toll Like Receptor 5 ◽  
Phosphatidylinositol 3

Background. Bacterial flagellin triggers inflammation in mammalian cells via Toll-like receptor (TLR) 5. Release of the chemokine IL-8 in response to flagellin involves NF-κB, p38 MAP kinase, and phosphatidylinositol 3-kinase (PI3K). However, PI3K has been reported to be either pro- or anti-inflammatory in different model systems. We hypothesized that this could be due to different activities of the p110αandβisoforms of PI3K.Results. PI3K and Akt were rapidly activated in Caco-2 colon carcinoma cells by flagellin. Using a plasmid-based shRNA delivery system and novel p110 isoform-specific inhibitors, we found that flagellin-induced IL-8 production was dependent on both p110αand p110β. However in the mouse, inhibition of p110βbut not p110αreduced the increase of serum IL-6 levels induced by intraperitoneal injection of flagellin.Conclusions. These data demonstrate that the p110αandβisoforms of class IA PI3K are both required for the proinflammatory response to flagellin.

scholarly journals PtdIns(4,5)P2 signaling regulates ATG14 and autophagy

2016 ◽  
Vol 113 (39) ◽  
pp. 10896-10901 ◽  
Author(s):  
Xiaojun Tan ◽  
Narendra Thapa ◽  
Yihan Liao ◽  
Suyong Choi ◽  
Richard A. Anderson
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Significance ◽  
Mammalian Cells ◽  
Beclin 1 ◽  
Class Iii ◽  
Phosphatidylinositol 3 Kinase ◽  
Atg Proteins ◽  
Late Endosomes ◽  
Irradiation Resistance ◽  
Phosphatidylinositol 3

Autophagy is a regulated self-digestion pathway with fundamental roles in cell homeostasis and diseases. Autophagy is regulated by coordinated actions of a series of autophagy-related (ATG) proteins. The Barkor/ATG14(L)–VPS34 (a class III phosphatidylinositol 3-kinase) complex and its product phosphatidylinositol 3-phosphate [PtdIns(3)P] play key roles in autophagy initiation. ATG14 contains a C-terminal Barkor/ATG14(L) autophagosome-targeting sequence (BATS) domain that senses the curvature of PtdIns(3)P-containing membrane. The BATS domain also strongly binds PtdIns(4,5)P2, but the functional significance has been unclear. Here we show that ATG14 specifically interacts with type Iγ PIP kinase isoform 5 (PIPKIγi5), an enzyme that generates PtdIns(4,5)P2 in mammalian cells. Autophagosomes have associated PIPKIγi5 and PtdIns(4,5)P2 that are colocalized with late endosomes and the endoplasmic reticulum. PtdIns(4,5)P2 generation at these sites requires PIPKIγi5. Loss of PIPKIγi5 results in a loss of ATG14, UV irradiation resistance-associated gene, and Beclin 1 and a block of autophagy. PtdIns(4,5)P2 binding to the ATG14–BATS domain regulates ATG14 interaction with VPS34 and Beclin 1, and thus plays a key role in ATG14 complex assembly and autophagy initiation. This study identifies an unexpected role for PtdIns(4,5)P2 signaling in the regulation of ATG14 complex and autophagy.

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

2008 ◽  
Vol 183 (3) ◽  
pp. 513-526 ◽  
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.

scholarly journals Role for phosphatidylinositol 3-kinase in the sorting and transport of newly synthesized lysosomal enzymes in mammalian cells.

1995 ◽  
Vol 130 (4) ◽  
pp. 781-796 ◽  
Author(s):  
W J Brown ◽  
D B DeWald ◽  
S D Emr ◽  
H Plutner ◽  
W E Balch
Keyword(s):  
Cathepsin D ◽  
Mammalian Cells ◽  
Lysosomal Enzymes ◽  
Secretory Pathway ◽  
Kinase Inhibitors ◽  
Phosphatidylinositol 3 Kinase ◽  
Yeast Saccharomyces Cerevisiae ◽  
The Media ◽  
Golgi Network ◽  
Enzyme Delivery

Previous work with the yeast Saccharomyces cerevisiae has demonstrated a role for a phosphatidylinositol-specific PI 3-kinase, the product of the VPS34 gene, in the targeting of newly synthesized proteins to the vacuole, an organelle functionally equivalent to mammalian lysosomes (Schu, P. V., K. Takegawa, M. J. Fry, J. H. Stack, M. D. Waterfield, and S. D. Emr. 1993. Science [Wash. DC]. 260:88-91). The activity of Vps34p kinase is significantly reduced by the PI 3-kinase inhibitors wortmannin, a fungal metabolite, and LY294002, a quercetin analog (Stack, J. H., and S. D. Emr. 1994. J. Biol. Chem. 269:31552-31562). We show here that at concentrations which inhibit VPS34-encoded PI 3-kinase activity, wortmannin also inhibits the processing and delivery of newly synthesized cathepsin D to lysosomes in mammalian cells with half-maximal inhibition of delivery occurring at 100 nM wortmannin. As a result of wortmannin action, newly synthesized, unprocessed cathepsin D is secreted into the media. Moreover, after accumulation in the trans-Golgi network (TGN) at 20 degrees C, cathepsin D was rapidly missorted to the secretory pathway after addition of wortmannin and shifting to 37 degrees C. At concentrations that inhibited lysosomal enzyme delivery, both wortmannin and LY294002 caused a highly specific dilation of mannose 6-phosphate receptor (M6PR)-enriched vesicles of the prelysosome compartment (PLC), which swelled to approximately 1 micron within 15 min after treatment. With increasing time, the inhibitors caused a significant yet reversible change in M6PR distribution. By 3 h of treatment, the swollen PLC vacuoles were essentially depleted of receptors and, in addition, there was a fourfold loss of receptors from the cell surface. However, M6PRs were still abundant in the TGN. These results are most consistent with the interpretation that PI 3-kinase regulates the trafficking of lysosomal enzymes by interfering with a M6PR-dependent sorting event in the TGN. Moreover, they provide evidence that trafficking of soluble hydrolases to mammalian lysosomes and yeast vacuoles rely on similar regulatory mechanisms.

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