Back to Search
Start Over
Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains.
- Source :
-
Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2021 Mar 02; Vol. 118 (9). - Publication Year :
- 2021
-
Abstract
- Possible segregation of plasma membrane (PM) phosphoinositide metabolism in membrane lipid domains is not fully understood. We exploited two differently lipidated peptide sequences, L10 and S15, to mark liquid-ordered, cholesterol-rich (L <subscript>o</subscript> ) and liquid-disordered, cholesterol-poor (L <subscript>d</subscript> ) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich L <subscript>o</subscript> phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor L <subscript>d</subscript> phases. The fluorescent protein markers were used as Förster resonance energy transfer (FRET) pairs in intact cells. An increase of homologous FRET between L10 probes showed that depleting membrane cholesterol shrank L <subscript>o</subscript> domains and enlarged L <subscript>d</subscript> domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged L <subscript>o</subscript> and shrank L <subscript>d</subscript> Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5) P <subscript>2</subscript> ] and phosphatidylinositol 4-phosphate (PtdIns4 P ) are present in both L <subscript>o</subscript> and L <subscript>d</subscript> domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5) P <subscript>2</subscript> and PtdIns4 P more rapidly and produced diacylglycerol (DAG) more rapidly in L <subscript>o</subscript> than in L <subscript>d</subscript> Further, PtdIns(4,5) P <subscript>2</subscript> was restored more rapidly in L <subscript>o</subscript> than in L <subscript>d</subscript> Thus destruction and restoration of PtdIns(4,5) P <subscript>2</subscript> are faster in L <subscript>o</subscript> than in L <subscript>d</subscript> This suggests that L <subscript>o</subscript> is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4 P pathway. The significant kinetic differences of lipid depletion and restoration also mean that exchange of lipids between these domains is much slower than free diffusion predicts.
- Subjects :
- Bacterial Proteins genetics
Bacterial Proteins metabolism
Cell Line, Transformed
Cholesterol metabolism
Diffusion
Diglycerides metabolism
Fluorescence Resonance Energy Transfer
Gene Expression
Genes, Reporter
Green Fluorescent Proteins genetics
Green Fluorescent Proteins metabolism
HEK293 Cells
Humans
Kinetics
Lipoylation
Luminescent Proteins genetics
Luminescent Proteins metabolism
Membrane Lipids metabolism
Peptides genetics
Receptors, Muscarinic genetics
Receptors, Muscarinic metabolism
Type C Phospholipases genetics
Type C Phospholipases metabolism
Unilamellar Liposomes metabolism
Membrane Microdomains metabolism
Peptides metabolism
Phosphatidylinositol 4,5-Diphosphate metabolism
Phosphatidylinositol Phosphates metabolism
Protein Processing, Post-Translational
Subjects
Details
- Language :
- English
- ISSN :
- 1091-6490
- Volume :
- 118
- Issue :
- 9
- Database :
- MEDLINE
- Journal :
- Proceedings of the National Academy of Sciences of the United States of America
- Publication Type :
- Academic Journal
- Accession number :
- 33619111
- Full Text :
- https://doi.org/10.1073/pnas.2025343118