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Diversity of raft-like domains in late endosomes.

Sobo K, Chevallier J, Parton RG, Gruenberg J, van der Goot FG - PLoS ONE (2007)

Bottom Line: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking.Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.Implications of these findings for late endosomal functions are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT

Background: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains.

Methodology/principal findings: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.

Conclusions/significance: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.

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Related in: MedlinePlus

Distribution of lipid metabolism-related proteins and raft markers in late endosomes.Late endosomes were purified from BHK cells and submitted to sub-organellar fractionation after breaking the organelle by cycles of freezing and thawing followed by sucrose density gradients. 12 fractions were collected from the top and analyzed for the presence of LBPA using an ELISA assay (A) or by SDS-PAGE followed by Western blotting for the presence NPC1, MLN64, flotillin-1 and ApoE (B). GPI-anchored proteins were detected by aerolysin overlay (B).
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pone-0000391-g004: Distribution of lipid metabolism-related proteins and raft markers in late endosomes.Late endosomes were purified from BHK cells and submitted to sub-organellar fractionation after breaking the organelle by cycles of freezing and thawing followed by sucrose density gradients. 12 fractions were collected from the top and analyzed for the presence of LBPA using an ELISA assay (A) or by SDS-PAGE followed by Western blotting for the presence NPC1, MLN64, flotillin-1 and ApoE (B). GPI-anchored proteins were detected by aerolysin overlay (B).

Mentions: We have previously shown that the internal membranes of late endosomes, which contain high amounts of LBPA [48], can be separated from the limiting membrane, after gentle mechanical disruption (by freeze thawing) of the organelle, followed by a continuous sucrose gradient [49]. Using this sub-organellar fractionation protocol, we analyzed the distribution of flotillin-1 and GPI-anchored proteins as well as that of three other proteins involved in cholesterol metabolism: ApoA1– an LDL apoprotein, MLN64 – a late endosomal steroidogenic acute regulatory protein (StAR) domain containing protein involved in sterol trafficking [58] and NPC1 – the Niemann Pick type C 1 protein involved in lipid trafficking [59]. GPI-anchored proteins co-fractionated with LBPA (Fig 4A), which was quantified by ELISA [49], and were mainly found in fractions 4 and 5 (Fig. 4B), indicating that these contained predominantly the intralumenal membranes of late endosomes. As expected, ApoA1, originating from internalized LDL particles, was also concentrated in fraction 4. In contrast, flotillin-1, MLN64 and NPC1 were all found in fractions 6 to 9 which also contain the limiting membrane marker Lamp1 [49]. These findings are in good agreement with electron microscopy studies in which MLN64 was found to be restricted to the limiting membrane of late endosomes [60]. These data altogether indicate that MLN64 and NPC1, which are both involved in sterol trafficking, localize to the limiting membrane.


Diversity of raft-like domains in late endosomes.

Sobo K, Chevallier J, Parton RG, Gruenberg J, van der Goot FG - PLoS ONE (2007)

Distribution of lipid metabolism-related proteins and raft markers in late endosomes.Late endosomes were purified from BHK cells and submitted to sub-organellar fractionation after breaking the organelle by cycles of freezing and thawing followed by sucrose density gradients. 12 fractions were collected from the top and analyzed for the presence of LBPA using an ELISA assay (A) or by SDS-PAGE followed by Western blotting for the presence NPC1, MLN64, flotillin-1 and ApoE (B). GPI-anchored proteins were detected by aerolysin overlay (B).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1851096&req=5

pone-0000391-g004: Distribution of lipid metabolism-related proteins and raft markers in late endosomes.Late endosomes were purified from BHK cells and submitted to sub-organellar fractionation after breaking the organelle by cycles of freezing and thawing followed by sucrose density gradients. 12 fractions were collected from the top and analyzed for the presence of LBPA using an ELISA assay (A) or by SDS-PAGE followed by Western blotting for the presence NPC1, MLN64, flotillin-1 and ApoE (B). GPI-anchored proteins were detected by aerolysin overlay (B).
Mentions: We have previously shown that the internal membranes of late endosomes, which contain high amounts of LBPA [48], can be separated from the limiting membrane, after gentle mechanical disruption (by freeze thawing) of the organelle, followed by a continuous sucrose gradient [49]. Using this sub-organellar fractionation protocol, we analyzed the distribution of flotillin-1 and GPI-anchored proteins as well as that of three other proteins involved in cholesterol metabolism: ApoA1– an LDL apoprotein, MLN64 – a late endosomal steroidogenic acute regulatory protein (StAR) domain containing protein involved in sterol trafficking [58] and NPC1 – the Niemann Pick type C 1 protein involved in lipid trafficking [59]. GPI-anchored proteins co-fractionated with LBPA (Fig 4A), which was quantified by ELISA [49], and were mainly found in fractions 4 and 5 (Fig. 4B), indicating that these contained predominantly the intralumenal membranes of late endosomes. As expected, ApoA1, originating from internalized LDL particles, was also concentrated in fraction 4. In contrast, flotillin-1, MLN64 and NPC1 were all found in fractions 6 to 9 which also contain the limiting membrane marker Lamp1 [49]. These findings are in good agreement with electron microscopy studies in which MLN64 was found to be restricted to the limiting membrane of late endosomes [60]. These data altogether indicate that MLN64 and NPC1, which are both involved in sterol trafficking, localize to the limiting membrane.

Bottom Line: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking.Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.Implications of these findings for late endosomal functions are discussed.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland.

ABSTRACT

Background: Late endosomes, the last sorting station in the endocytic pathway before lysosomes, are pleiomorphic organelles composed of tubular elements as well as vesicular regions with a characteristic multivesicular appearance, which play a crucial role in intracellular trafficking. Here, we have investigated whether, in addition to these morphologically distinguishable regions, late endosomal membranes are additionally sub-compartmentalized into membrane microdomains.

Methodology/principal findings: Using sub-organellar fractionation techniques, both with and without detergents, combined with electron microscopy, we found that both the limiting membrane of the organel and the intraluminal vesicles contain raft-type membrane domains. Interestingly, these differentially localized domains vary in protein composition and physico-chemical properties.

Conclusions/significance: In addition to the multivesicular organization, we find that late endosomes contain cholesterol rich microdomains both on their limiting membrane and their intraluminal vesicles that differ in composition and properties. Implications of these findings for late endosomal functions are discussed.

Show MeSH
Related in: MedlinePlus