Abstract
BackgroundLate 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 FindingsUsing 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/SignificanceIn 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.
Highlights
The general view of the cellular plasma membrane has evolved, over the last 20 years, from that of a homogeneous arrangement of lipids with embedded proteins towards that of a mosaic of microdomains, each having a specific lipid and protein composition [1]
We have previously shown that detergent resistant membranes (DRMs) can be isolated from late endosomes [29] purified from baby hamster kidney (BHK) cells using a well-established subcellular fractionation protocol [29,47]
To test whether late endosomal DRMs are sensitive to cholesterol affecting drugs, an important criterion for being a raft-like domain [4], we treated late endosomes with either the cholesterol clustering agent saponin [53,54] or the cholesterol binding compound filipin [38]
Summary
The general view of the cellular plasma membrane has evolved, over the last 20 years, from that of a homogeneous arrangement of lipids with embedded proteins towards that of a mosaic of microdomains, each having a specific lipid and protein composition [1]. 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. These differentially localized domains vary in protein composition and physico-chemical properties. 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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