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
SN - 0027-8424
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
IS - 26
ER -