The Ligand and Membrane-binding Behaviour of the Phosphatidylinositol Transfer Proteins (PITPa & PITPb)
Abstract
Human Class I phosphatidylinositol transfer proteins (PITPs) exists in two forms: PITPα
and PITPβ. PITPs are believed to be lipid transfer proteins based on their capacity to transfer
either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments
in vitro. In Drosophila, the PITP domain is found to be part of a multi-domain protein named
retinal degeneration B (RdgBα). The PITP domain of RdgBα shares 40 % sequence identity
with PITPα and has been shown to possess PI and PC binding and transfer activity.
The detailed molecular mechanism of ligand transfer by the human PITPs and the
Drosophila PITP domain remains to be fully established. Here, we investigated the membrane
interactions of these proteins using dual polarization interferometry (DPI). DPI is a technique
that measures protein binding affinity to a flat immobilized lipid bilayer. In addition, we also
measured how quickly these proteins transfer their ligands to lipid vesicles using a fluorescence
resonance energy transfer (FRET)-based assay.
DPI investigations suggest that PITPβ had a two-fold higher affinity for membranes
compared to PITPα. This was reflected by a four-fold faster ligand transfer rate for PITPβ in
comparison to PITPα as determined by the FRET assay. Interestingly, DPI analysis also
demonstrated that PI-bound human PITPs have lower membrane affinity compared to PC-bound
PITPs.
In addition, the FRET studies demonstrated the significance of membrane curvature in
the ligand transfer rate of PITPs. The ligand transfer rate was higher when the accepting vesicles
were highly curved. Furthermore, when the accepting vesicles contained phosphatidic acid (PA)
which have smaller head groups, the transfer rate increased. In contrast, when the accepting
vesicles contained phosphoinositides which have larger head groups, the transfer rate was
diminished. However, PI, the favorite ligand of PITPs, or the presence of anionic lipids did not
appear to influence the ligand transfer rate of PITPs.
Both DPI and FRET examinations revealed that the PITP domain of RdgBα was able to
bind to membranes. However, the RdgBα PITP domain appears to be a poor binder and
transporter of PC.