Effects of Cholesterol Depletion and Increased Lipid Unsaturation on the Properties of Endocytic Membranes

Mingming Hao,Sushmita Mukherjee,Yu Sun,Frederick R Maxfield

doi:10.1074/jbc.m309793200

Mingming Hao, Sushmita Mukherjee + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m309793200

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Abstract

Lipid analogs with dialkylindocarbocyanine (DiI) head groups and short or unsaturated hydrocarbon chains (e.g. DiIC(12) and FAST DiI) enter the endocytic recycling compartment efficiently, whereas lipid analogs with long, saturated tails (e.g. DiIC(16) and DiIC(18)) are sorted out of this pathway and targeted to the late endosomes/lysosomes (Mukherjee, S., Soe, T. T., and Maxfield, F. R. (1999) J. Cell Biol. 144, 1271-1284). This differential trafficking of lipid analogs with the same polar head group was interpreted to result from differential partitioning to different types of domains with varying membrane order and/or curvature. Here we investigate the system further by monitoring the trafficking behavior of these lipid analogs under conditions that alter domain properties. There was a marked effect of cholesterol depletion on the cell-surface distribution and degree of internalization of the lipid probes. Furthermore, instead of going to the late endosomes/lysosomes as in control cells, long chain DiI analogs, such as DiIC(16), were sorted to the recycling pathway in cholesterol-depleted cells. We confirmed that this difference was due to a change in overall membrane properties, and not cholesterol levels per se, by utilizing a Chinese hamster ovary cell line that overexpressed transfected stearoyl-CoA desaturase 1, a rate-limiting enzyme in the production of monounsaturated fatty acids. These cells have a decrease in membrane order because they contain a much larger fraction of unsaturated fatty acids. These cells showed alteration of DiI trafficking very similar to cholesterol-depleted cells. By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes.

Highlights

Lipids and proteins associated with the cell surface vary in their lateral and transbilayer distribution, as well as the rate at which they are internalized from the plasma membrane
By using cold Triton X-100 extractability of different lipids as a criterion to determine the membrane properties of intracellular organelles, we found that the endocytic recycling compartment has abundant detergent-resistant membranes, in contrast to the late endosomes and lysosomes
It has been shown previously [29] that cholesterol depletion severely inhibits the internalization of TfR from the cell surface. Under these conditions, most of the Tf remains at the cell surface, and the bright cell surface fluorescence impedes the detection of the central endocytic recycling compartment (ERC) fluorescence

Summary

EXPERIMENTAL PROCEDURES

All fluorescent probes and anti-Alexa 488 were obtained from Molecular Probes Inc. (Eugene, OR). All fluorescent probes and anti-Alexa 488 were obtained from Molecular Probes Inc. It was iron-loaded and passed through a Sephacryl S-300 gel filtration system as described previously [18]. Alexa 488 was conjugated to the iron-loaded Tf following manufacturers’ instructions. Labeled transferrin was dialyzed thoroughly to remove the unbound dye. Monoclonal antibody against DAF was provided by Dr S. Tomlinson (Medical University of South Carolina) [19]. DHE-loaded M␤CD was prepared as described previously [17]. DiI-LDL was a gift from Dr Ira Tabas (Columbia University, New York). Lipid analogs and free fatty acids were transferred as monomers from fatty acid-free BSA carriers [1].

The abbreviations used are

RESULTS

DISCUSSION