Separation and Characterization of Late Endosomal Membrane Domains

Kobayashi, Toshihide; Beuchat, Marie-HÃ©lÃ ̈ne; Chevallier, Julien; Makino, Asami; Mayran, Nathalie; Escola, Jean-Michel; Lebrand, Cécile; Cosson, Pierre; Kobayashi, Tetsuyuki; Grüenberg, Jean

doi:10.1074/jbc.m202838200

Cited by 358 publications

(417 citation statements)

References 38 publications

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“…DRM preparations selectively enrich for cholesterol-rich membranes comprised of plasma membrane DRMs and late endosomal DRMs [23]. In this study we show that deficiency of CLN3 protein results in less buoyant DRMs (Fig.…”

Section: Discussionsupporting

confidence: 54%

A novel role of the Batten disease gene CLN3: Association with BMP synthesis

Hobert

Dawson

2007

Biochemical and Biophysical Research Communications

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Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) results from a deficiency of CLN3, a protein recently identified within detergent-resistant membranes (DRMs). To study the function of CLN3 within these domains we isolated DRMs from control and JNCL-brain and noted that JNCL-derived DRMs are less buoyant than control. Analysis of DRM phospholipids derived from JNCL-brain revealed a reduction of bis(monoacylglycerol)phosphate. Metabolic labeling of JNCL-fibroblasts demonstrated a reduction in the synthesis of bis(monoacylglycerol)phosphate which was restored following complementation with wild-type-CLN3, substantiating our initial observation in brain. Metabolic labeling of cell lines overexpressing wild-type-CLN3 resulted in increased bis (monoacylglycerol)phosphate synthesis, while overexpression of mutant CLN3-L170P decreased bis(monoacylglycerol)phosphate synthesis. These data illustrate a new finding, a strong correlation between CLN3 protein expression and synthesis of bis(monoacylglycerol)phosphate.

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Section: Discussionsupporting

confidence: 54%

A novel role of the Batten disease gene CLN3: Association with BMP synthesis

Hobert

Dawson

2007

Biochemical and Biophysical Research Communications

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“…(Supplemental data for this article is available online at the American Journal of Physiology-Lung Cellular and Molecular Physiology website.) The smeared appearance of the immunoblot signal reflects high glycosylation states of LAMP3 protein (16). On the other hand, the levels of GRP78, a marker of endoplasmic reticulum, were similar in the five cell lines.…”

Section: Subcellular Localization and Glycosylation Of Abca3-gfp And mentioning

confidence: 79%

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

Matsumura¹,

Ban²,

Inagaki³

2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[ 32 P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

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“…However, we find that all endosomes that incorporated nanogold into newly formed lumenal vesicles in vitro also contained endocytosed EGF receptor and, conversely, that the EGF receptor is itself sorted into lumenal vesicles in a process controlled by the opposite actions of Alix and Tsg101, like HPTS incorporation. An alternative explanation comes from the observations that multivesicular endosomes contain more than one type of lumenal vesicles (Gillooly et al, 2000;Kobayashi et al, 2002;Sobo et al, 2007a), raising the possibility that some are involved in the transport of signaling receptors to the lysosomes, whereas others undergo fusion at the limiting membrane. If so, intralumenal mechanisms must control the fate of intralumenal vesicles, back-fusion or degradation, including perhaps LBPA via its effector Alix, because Alix knockdown inhibits back-fusion (Abrami et al, 2004;Le Blanc et al, 2005) but not EGF receptor degradation (Schmidt et al, 2004;Cabezas et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

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

Falguières

Luyet

Bissig

et al. 2008

MBoC

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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., ...

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Separation and Characterization of Late Endosomal Membrane Domains

Cited by 358 publications

References 38 publications

A novel role of the Batten disease gene CLN3: Association with BMP synthesis

A novel role of the Batten disease gene CLN3: Association with BMP synthesis

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

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

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