Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles

CDP-diacylglycerol phospholipid synthesis in detergent-soluble, non-raft, membrane microdomains of the endoplasmic reticulum

“…This led us to modify our membrane fractionation scheme and to carry out the experiment in the absence of carbonate at neutral pH while retaining the probe sonication step, which we have shown previously to result in the formation of membrane vesicles in the 50-250 nm diameter size range ( 32,48,49 ). Using this approach, we observed almost identical gradient distributions of CDP-DAG and PI generation ( Fig.…”

“…The density shift of these membranes apparent following carbonate treatment indicates that peripherally associated proteins account for the lack of buoyancy in neutral pH buffer ( 51 ). This differs from cholesterol-rich membranes at other cellular sites, such as the trans -Golgi network, which tend to be intrinsically buoyant due to their relatively low protein-to-lipid ratio ( 56 ). The low abundance of cholesterol at the ER, which is a tightly regulated process ( 57 ), and the exclusion of cholesterol from model membranes containing unsaturated lipid side chains ( 58,59 ) further argue against a predominant role for classical lipid raft domains in the organization of phospholipid synthesis.…”

“…We have shown on a number of previous occasions that the sonication procedure we use to disrupt the ER membranes gives rise to membrane vesicles in the size range of 60-200 nm diameter ( 32,48,49 ); hence, the ability "non-raft" phospholipid-rich membrane domains, the existence of which was fi rst postulated by Shaikh and Edidin ( 35 ). To the best of our knowledge, this is the fi rst experimental evidence that such membrane domains exist in cells and furthermore, our results indicate that these domains are an important site for cellular phospholipid synthesis.…”

El metabolismo del colesterol: cada vez más complejo

“…La razón colesterol/proteínas en las membranas de células de mamíferos varía notablemente. El esterol es distribuido heterogénea-mente entre las distintas membranas celulares siendo más abundante en la membrana plasmáti-ca y del Golgi (Waugh et al, 2011). En la membrana plasmática el colesterol constituye alrededor del 80 -90% del colesterol celular total y entre el 20-25% del total de moléculas lipídicas (Ikonen, 2008).…”

“…En la membrana plasmática el colesterol constituye alrededor del 80 -90% del colesterol celular total y entre el 20-25% del total de moléculas lipídicas (Ikonen, 2008). El colesterol es abundante, especialmente en el trans-Golgi y en los compartimientos endocí-ticos de reciclaje (Waugh et al, 2011). En contraste, tanto en la mitocondria como en el RE los niveles de colesterol son muy bajos.…”