Human VPS34 is required for internal vesicle formation within multivesicular endosomes

Futter, Clare E.; Collinson, Lucy M.; Backer, Jonathan M.; Hopkins, Colin R.

doi:10.1083/jcb.200108152

Cited by 227 publications

(230 citation statements)

References 59 publications

Supporting

Mentioning

208

Contrasting

Unclassified

Order By: Relevance

“…5). This finding is consistent with previous studies on the effects of PI 3-kinase inhibitors on the degradation of other signaling receptors, such as the PDGF receptor [16] and the EGF receptor [25,26] There are at least three PI 3-kinases in mammalian cells, and the most commonly used PI 3-kinase inhibitors act on all three [27]. Evidence from other studies implicates the type III PI 3-kinase hVps34 in controlling traffic to lysosomes [28,29].…”

Section: Discussionsupporting

confidence: 91%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

View full text Add to dashboard Cite

Phosphatidylinositol 3-kinase inhibitors have been shown to affect the endocytosis or subsequent intracellular sorting in various receptor systems. Agonist-activated β 2 -adrenergic receptors undergo desensitization by mechanisms that include the phosphorylation, endocytosis and degradation of receptors. Following endocytosis, most internalized receptors are sorted to the cell surface, but some proportion is sorted to lysosomes for degradation. It is not known what governs the ratio of receptors that recycle versus receptors that undergo degradation. To determine if phosphatidylinositol 3-kinases regulate β 2 -adrenergic receptor trafficking, HEK293 cells stably expressing these receptors were treated with the phosphatidylinositol 3-kinase inhibitors LY294002 or wortmannin. We then studied agonist-induced receptor endocytosis and postendocytic sorting, including recycling and degradation of the internalized receptors. Both inhibitors amplified the internalization of receptors after exposure to the β-agonist isoproterenol, which was attributable to the sorting of a significant fraction of receptors to an intracellular compartment from which receptor recycling did not occur. The initial rate of β 2 -adrenergic receptor endocytosis and the default rate of receptor recycling were not significantly altered. During prolonged exposure to agonist, LY294002 slowed the degradation rate of β 2 -adrenergic receptors and caused the accumulation of receptors within rab7-positive vesicles. These results suggest that phosphatidylinositol 3-kinase inhibitors (1) cause a misrouting of β 2 -adrenergic receptors into vesicles that are neither able to efficiently recycle to the surface nor sort to lysosomes, and (2) delays the movement of receptors from late endosomes to lysosomes.

show abstract

Section: Discussionsupporting

confidence: 91%

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Awwad

Iyer

Rosenfeld

et al. 2007

Experimental Cell Research

View full text Add to dashboard Cite

show abstract

“…The process is quantified by measuring the incorporation of the fluorescent solute HPTS within newly formed vesicles that budded into the endosome lumen. These data indicate that intralumenal vesicles may represent Ϸ10% of the total volume of late endosomes in BHK cells , a value that fits nicely with electron microscopy observations by others (Futter et al, 2001;Murk et al, 2003;Mari et al, 2008) and us (Le Blanc et al, 2005). Indeed, a multivesicular endosome of 0.4 -0.5-m diameter contains Ϸ30 -60 vesicles (50 -60-nm diameter), representing in total Ϸ8 -15% of the organelle volume.…”

Section: Discussionsupporting

confidence: 89%

“…Evidence shows that this PtdIns3P-, Hrs-, and ESCRT-based sorting mechanism is coupled to the membrane invagination process. The number of intralumenal vesicles within endosomes is decreased after PtdIns 3-kinase inhibition (Reaves et al, 1996;Fernandez-Borja et al, 1999;Futter et al, 2001), and after Hrs knockout in Drosophila melanogaster (Lloyd et al, 2002) or knockdown in mammalian cells (Bache et al, 2003b;Razi and Futter, 2006). Presumably, Hrs, by initiating ES-CRT recruitment, is indirectly responsible for intralumenal vesicle formation (Bache et al, 2003a).…”

mentioning

confidence: 99%

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Falguières

Luyet

Bissig

et al. 2008

MBoC

View full text Add to dashboard Cite

Endosomes along the degradation pathway leading to lysosomes accumulate membranes in their lumen and thus exhibit a characteristic multivesicular appearance. These lumenal membranes typically incorporate down-regulated EGF receptor destined for degradation, but the mechanisms that control their formation remain poorly characterized. Here, we describe a novel quantitative biochemical assay that reconstitutes the formation of lumenal vesicles within late endosomes in vitro. Vesicle budding into the endosome lumen was time-, temperature-, pH-, and energy-dependent and required cytosolic factors and endosome membrane components. Our light and electron microscopy analysis showed that the compartment supporting the budding process was accessible to endocytosed bulk tracers and EGF receptor. We also found that the EGF receptor became protected against trypsin in our assay, indicating that it was sorted into the intraendosomal vesicles that were formed in vitro. Our data show that the formation of intralumenal vesicles is ESCRT-dependent, because the process was inhibited by the K173Q dominant negative mutant of hVps4. Moreover, we find that the ESCRT-I subunit Tsg101 and its partner Alix control intralumenal vesicle formation, by acting as positive and negative regulators, respectively. We conclude that budding of the limiting membrane toward the late endosome lumen, which leads to the formation of intraendosomal vesicles, is controlled by the positive and negative functions of Tsg101 and Alix, respectively. INTRODUCTIONIn eukaryotic cells, molecules of the plasma membrane, ligands and solutes are internalized into early endosomes, from where they can be recycled back to the plasma membrane, transported to the trans-Golgi network, or targeted to late endosomes and lysosomes (Gruenberg, 2001;Maxfield and McGraw, 2004;Mayor and Pagano, 2007). The latter pathway is followed in particular by ubiquitinated signaling receptors that need to be down-regulated. These receptors are incorporated into invaginations of the early endosomal membrane that form within their lumen (Hurley and Emr, 2006), thus giving rise to nascent multivesicular bodies (MVBs) or endosomal carrier vesicles (ECVs; Gruenberg and Stenmark, 2004;Piper and Katzmann, 2007), which function as intermediates between early and late endosomes. Eventually, intralumenal vesicles are delivered to lysosomes, where they are degraded together with their receptor cargo.Major progress has been made in our understanding of the molecular mechanisms that control the sorting of ubiquitinated receptors, in particular the epidermal growth factor (EGF) receptor (Hurley and Emr, 2006;Slagsvold et al., 2006;Piper and Katzmann, 2007;Williams and Urbe, 2007). These receptors interact via the ubiquitin moiety with hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs), which in turn binds clathrin and phosphatidyl-inositol-3-phosphate (PtdIns3P), thereby concentrating the receptor in early endosomal membrane domains (Raiborg et al., 2001;Urbe et al., 2003;Raiborg et al., ...

show abstract

“…45,46 PI3K inhibition thus limits MVB maturation. [46][47][48][49] WT and PKD1 − / − 3 − / − GFP-CD63 + DT40 cells were incubated alone or with the PI3K inhibitor LY294002. Inhibitor-treated WT DT40 cells formed a cluster of enlarged, ring-shaped CD63 + vesicles similar to those found constitutively in PKD1 − / − 3 − / − DT40 cells (Figure 6a).…”

Section: Resultsmentioning

confidence: 99%

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

et al. 2015

View full text Add to dashboard Cite

Multivesicular bodies (MVBs) are endocytic compartments that enclose intraluminal vesicles (ILVs) formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these ILV contain Fas ligand (FasL) and are secreted as 'lethal exosomes' following activation-induced fusion of the MVB with the plasma membrane. Diacylglycerol (DAG) and diacylglycerol kinase α (DGKα) regulate MVB maturation and polarized traffic, as well as subsequent secretion of pro-apoptotic exosomes, but the molecular basis underlying these phenomena remains unclear. Here we identify protein kinase D (PKD) family members as DAG effectors involved in MVB genesis and secretion. We show that the inducible secretion of exosomes is enhanced when a constitutively active PKD1 mutant is expressed in T lymphocytes, whereas exosome secretion is impaired in PKD2-deficient mouse T lymphoblasts and in PKD1/3-null B cells. Analysis of PKD2-deficient T lymphoblasts showed the presence of large, immature MVB-like vesicles and demonstrated defects in cytotoxic activity and in activation-induced cell death. Using pharmacological and genetic tools, we show that DGKα regulates PKD1/2 subcellular localization and activation. Our studies demonstrate that PKD1/2 is a key regulator of MVB maturation and exosome secretion, and constitutes a mediator of the DGKα effect on MVB secretory traffic. Exosomes are nanovesicles that form as intraluminal vesicles (ILVs) inside multivesicular bodies (MVBs) and are then secreted by numerous cell types. 1 ILVs are generated by inward budding of late endosome limiting membrane in a precisely regulated maturation process. 2,3 Two main pathways are involved in MVB maturation. 4,5 In addition to the ESCRT (endosomal complex required for traffic) proteins, 6 there is increasing evidence that lipids such as lyso-bisphosphatidic acid (LBPA), 7 ceramides 8 and diacylglycerol (DAG) 9 contribute to this membrane invagination process.Exosomes participate in many biological processes related to T-cell receptor (TCR)-triggered immune responses, including T lymphocyte-mediated cytotoxicity and activationinduced cell death (AICD), antigen presentation and intercellular miRNA exchange. [10][11][12][13][14][15] The discovery of exosome involvement in these responses increased interest in the regulation of exosome biogenesis and secretory traffic, with special attention to the contribution of lipids such as ceramide and DAG, as well as DAG-binding proteins. 14,16-21 These studies suggest that positive and negative DAG regulators may control secretory traffic. By transforming DAG into phosphatidic acid (PA), diacylglycerol kinase α (DGKα) is essential for the negative control of DAG function in T lymphocytes. 22 DGKα translocates transiently to the T-cell membrane after human muscarinic type 1 receptor (HM1R) triggering or to the immune synapse (IS) after TCR stimulation; at these subcellular locations, DGKα acts as a negative modulator of phospholipase C (PLC)-generated DAG. 23,24 The secretory vesicle pathway involves several DAGc...

show abstract

Human VPS34 is required for internal vesicle formation within multivesicular endosomes

Cited by 227 publications

References 59 publications

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

Inhibitors of phosphoinositide 3-kinase cause defects in the postendocytic sorting of β2-adrenergic receptors

In Vitro Budding of Intralumenal Vesicles into Late Endosomes Is Regulated by Alix and Tsg101

Protein kinase D1/2 is involved in the maturation of multivesicular bodies and secretion of exosomes in T and B lymphocytes

Contact Info

Product

Resources

About