The C‐terminal domain of perfringolysin O is an essential cholesterol‐binding unit targeting to cholesterol‐rich microdomains

Abstract

There is much evidence to indicate that cholesterol forms lateral membrane microdomains (rafts), and to suggest their important role in cellular signaling. However, no probe has been produced to analyze cholesterol behavior, especially cholesterol movement in rafts, in real time. To obtain a potent tool for analyzing cholesterol dynamics in rafts, we prepared and characterized several truncated fragments of θ‐toxin (perfringolysin O), a cholesterol‐binding cytolysin, whose chemically modified form has been recently shown to bind selectively to rafts. BIAcore and structural analyses demonstrate that the C‐terminal domain (domain 4) of the toxin is the smallest functional unit that has the same cholesterol‐binding activity as the full‐size toxin with structural stability. Cell membrane‐bound recombinant domain 4 was detected in the floating low‐density fractions and was found to be cofractionated with the raft‐associated protein Lck, indicating that recombinant domain 4 also binds selectively to cholesterol‐rich rafts. Furthermore, an enhanced green fluorescent protein‐domain 4 fusion protein stains membrane surfaces in a cholesterol‐dependent manner in living cells. Therefore, domain 4 of θ‐toxin is an essential cholesterol‐binding unit targeting to cholesterol in membrane rafts, providing a very useful tool for further studies on lipid rafts on cell surfaces and inside cells.

Latent infection membrane protein transmembrane FWLY is critical for intermolecular interaction, raft localization, and signaling

“…Tryptophan residues have been implicated in hydrophobic ring and Raft interactions; FWLY 38 -41 may mediate interactions with other ring residues in LMP1 or with cholesterol-rich microdomains (53)(54)(55)(56). The failure of LMP1AALA 38 -41 to associate with Raft and Barge membrane microdomains and to signal, supports the hypothesis that LMP1 transmembrane domain-mediated intermolecular aggregation in Raft or Barge microdomains is an important nucleation event for signaling.…”

Irreversible loss of membrane-binding activity of Listeria-derived cytolysins in non-acidic conditions: a distinct difference from allied cytolysins produced by other Gram-positive bacteria

“…It was demonstrated that at least the step of LLO binding to membrane cholesterol is affected by denaturation under neutral or alkaline pH conditions. CDCs have four functional domains, among which the C-terminal domain 4 is essential for the recognition and first binding of CDCs to target membrane (Rossjohn et al, 1997;Shimada et al, 2002). Several studies have suggested that the domain 4 of CDCs remains close to the membrane surface in the membrane-inserted oligomer without being embedded deeply in the bilayer (Heuck et al, 2000;Ramachandran et al, 2004).…”

“…This was because domain 4 is responsible for the membrane-binding activity of CDCs (Shimada et al, 2002) and the leucine residue at position 461 in domain 4 is implicated in the pH-dependent cytolytic activity of LLO (Glomski et al, 2002). To confirm this possibility, we constructed domain 4 preparations of LLO and PLY as representatives of pH-sensitive and pH-insensitive CDCs, respectively.…”

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

“…Finally, as a third approach to track intracellular cholesterol content, we monitored the distribution of the cholesterolbinding domain (D4) of perfringolysin O (Shimada et al, 2002), fused to mCherry (Abe et al, 2012). When expressed in hippocampal CA1 neurons, mCherry-D4 displayed a diffuse distribution with the presence of scattered fluorescence clusters ( Fig.…”