Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

Research output: Contribution to journalArticle

53 Citations (Scopus)

Abstract

Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study,we generate an 125I-labeledmutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to 125I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The 125I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines,we showthat this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

Original languageEnglish (US)
Pages (from-to)10580-10585
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume110
Issue number26
DOIs
StatePublished - Jun 25 2013

Fingerprint

Cholesterol
Cell Membrane
Lysosomes
Organelles
Membranes
Cholesterol Esters
Endoplasmic Reticulum
LDL Cholesterol
Clostridium perfringens theta-toxin
Hydrolysis
Lipids
Cell Line
oxidized low density lipoprotein

ASJC Scopus subject areas

  • General

Cite this

@article{96873d9bf03843e298d813204803b77c,
title = "Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells",
abstract = "Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study,we generate an 125I-labeledmutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to 125I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol{\%} of total lipids. This 35{\%} threshold is in striking contrast to the 5{\%} threshold previously found for PFO binding to endoplasmic reticulum membranes. The 125I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines,we showthat this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.",
author = "Akash Das and Goldstein, {Joseph L.} and Anderson, {Donald D.} and Brown, {Michael S.} and Arun Radhakrishnan",
year = "2013",
month = "6",
day = "25",
doi = "10.1073/pnas.1309273110",
language = "English (US)",
volume = "110",
pages = "10580--10585",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "26",

}

TY - JOUR

T1 - Use of mutant 125I-Perfringolysin O to probe transport and organization of cholesterol in membranes of animal cells

AU - Das, Akash

AU - Goldstein, Joseph L.

AU - Anderson, Donald D.

AU - Brown, Michael S.

AU - Radhakrishnan, Arun

PY - 2013/6/25

Y1 - 2013/6/25

N2 - Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study,we generate an 125I-labeledmutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to 125I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The 125I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines,we showthat this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

AB - Animal cells strictly control the distribution of cholesterol in their organelle membranes. This regulation requires an efficient machinery to transport insoluble cholesterol between organelles. In the present study,we generate an 125I-labeledmutant version of Perfringolysin O (PFO), a cholesterol-binding protein, and use it to measure cholesterol in the plasma membrane of intact cells. We also purify plasma membranes from the same cells, which allows us to directly relate cholesterol concentration to 125I-PFO binding. We show that cholesterol is organized in the plasma membrane in a manner that makes it inaccessible to PFO until its concentration exceeds a threshold of 35 mol% of total lipids. This 35% threshold is in striking contrast to the 5% threshold previously found for PFO binding to endoplasmic reticulum membranes. The 125I-PFO probe also proved useful in monitoring the movement of LDL-derived cholesterol from lysosomes to plasma membranes. Using three different mutant cell lines,we showthat this transport requires receptor-mediated uptake of LDL, hydrolysis of LDL-cholesteryl esters in lysosomes, and transfer of the liberated cholesterol to the plasma membrane.

UR - http://www.scopus.com/inward/record.url?scp=84879533612&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84879533612&partnerID=8YFLogxK

U2 - 10.1073/pnas.1309273110

DO - 10.1073/pnas.1309273110

M3 - Article

C2 - 23754385

AN - SCOPUS:84879533612

VL - 110

SP - 10580

EP - 10585

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 26

ER -