Compartmentalization of phosphatidylinositol 4,5-bisphosphate metabolism into plasma membrane liquid-ordered/raft domains

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 (Lo) and liquid-disordered, cholesterol-poor (Ld) domains of the PM, often called raft and nonraft domains, respectively. Imaging of the fluorescent labels verified that L10 segregated into cholesterol-rich Lo phases of cooled giant plasma-membrane vesicles (GPMVs), whereas S15 and the dye FAST DiI cosegregated into cholesterol-poor Ld 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 Lo domains and enlarged Ld domains, whereas a decrease of L10 FRET showed that adding more cholesterol enlarged Lo and shrank Ld. Heterologous FRET signals between the lipid domain probes and phosphoinositide marker proteins suggested that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and phosphatidylinositol 4-phosphate (PtdIns4P) are present in both Lo and Ld domains. In kinetic analysis, muscarinic-receptor-activated phospholipase C (PLC) depleted PtdIns(4,5)P2 and PtdIns4P more rapidly and produced diacylglycerol (DAG) more rapidly in Lo than in Ld. Further, PtdIns(4,5)P2 was restored more rapidly in Lo than in Ld. Thus destruction and restoration of PtdIns(4,5)P2 are faster in Lo than in Ld. This suggests that Lo is enriched with both the receptor G protein/PLC pathway and the PtdIns/PI4-kinase/PtdIns4P 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.

Striking a balance: PIP2 and PIP3 signaling in neuronal health and disease

“…Plasma membrane microdomains can be classified, on the basis of cholesterol abundance, into cholesterolrich, liquid-ordered (L o ) raft domains or cholesterol-poor, liquid-disordered (L d ) non-raft domains. Recent literature suggests that PIP 2 is present in both domains, however, it gets hydrolyzed by PLC faster, and is also restored more rapidly in cholesterol-rich (L o ) domain [102]. This compartmentalization of PIP 2 signaling seems to be conserved as it has been reported in plants membranes as well [103].…”

A palmitoylation code controls PI4KIIIα complex formation and PI(4,5)P2 homeostasis at the plasma membrane

“…PI(4,5)P2 is enriched in Lo-like regions of the PM [28,[40][41][42] , and some evidence points to its local synthesis in these membrane domains [28,43] . However, several studies have also detected substantial pools of PI(4,5)P2 in Ld-like membrane domains [28,40,42] . The mechanisms that lead to specific synthesis and enrichment of PI(4,5)P2 in Lo or Ld domains are not well understood.…”

“…The moderate Lo-preference of EFR3B also indicates that a substantial minority fraction of EFR3B is present in Ld-like domains at steadystate. Having pools of EFR3, the membrane anchor for all known PI4KIIIa complexes, in both Lo and Ld-pools could be functionally relevant, because PI(4,5)P2 is likely synthesized in both domains [28,40,42] , requiring recruitment of PI4KIIIα to both Lo-and Ld-like domains.…”

“…Because the sole shared component between these two PI4KIIIα complexes is EFR3, whose membrane anchoring requires a Cys-rich palmitoylation motif, we posited that differential localization within Lo-like and Ld-like regions of the PM may be a defining difference between Complexes I and II critical to their unique functional properties. PI(4,5)P2 is enriched in Lo-like regions of the PM [28,[40][41][42] , and some evidence points to its local synthesis in these membrane domains [28,43] . However, several studies have also detected substantial pools of PI(4,5)P2 in Ld-like membrane domains [28,40,42] .…”

“…Palmitoylation of Cys-rich motifs within proteins can mediate partitioning of such proteins to liquid-ordered like (Lo-like) membrane domains, which are enriched in lipids with saturated tails [27] . Interestingly, PI(4,5)P2 synthesis was recently proposed to occur in these domains [28] , though the mechanisms of Lo or the complementary liquid-disordered (Ld)-like membrane domain compartmentalization of PI(4,5)P2 synthesis are not fully understood.…”