Dynamics of lipid transfer by phosphatidylinositol transfer proteins in cells.
Of many lipid transfer proteins identified, all have been implicated in essential cellular processes, but the activity of none has been demonstrated in intact cells. Among these, phosphatidylinositol transfer proteins (PITP) are of particular interest as they can bind to and transfer phosphatidylinositol (PtdIns)--the precursor of important signalling molecules, phosphoinositides--and because they have essential functions in neuronal development (PITPalpha) and cytokinesis (PITPbeta). Structural analysis indicates that, in the cytosol, PITPs are in a 'closed' conformation completely shielding the lipid within them. But during lipid exchange at the membrane, they must transiently 'open'. To study PITP dynamics in intact cells, we chemically targeted their C95 residue that, although non-essential for lipid transfer, is buried within the phospholipid-binding cavity, and so, its chemical modification prevents PtdIns binding because of steric hindrance. This treatment resulted in entrapment of open conformation PITPs at the membrane and inactivation of the cytosolic pool of PITPs within few minutes. PITP isoforms were differentially inactivated with the dynamics of PITPbeta faster than PITPalpha. We identify two tryptophan residues essential for membrane docking of PITPs.
Shadan
Sadaf
S
Department of Cell and Developmental Biology, Lipid Signalling Group, University College London, London, UK.
Holic
Roman
R
Carvou
Nicolas
N
Ee
Patrick
P
Li
Michelle
M
Murray-Rust
Judith
J
Cockcroft
Shamshad
S
D015082:2,2'-Dipyridyl / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002522:Chlorocebus aethiops; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004220:Disulfides / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D016716:PC12 Cells; D010716:Phosphatidylinositols / Q000737:chemistry / Q000378:metabolism*; D049294:Phospholipid Transfer Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D021381:Protein Transport; D051381:Rats; D014162:Transfection; D014364:Tryptophan / Q000378:metabolism
D004220:Disulfides; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; C001105:2,2'-dipyridyl disulfide; D015082:2,2'-Dipyridyl; D014364:Tryptophan; D005033:Ethylmaleimide