Functional reconstitution of the human placental transferrin receptor into phospholipid bilayers leads to long tubular structures proceeding from the vesicle surface.
We have reconstituted the human placental transferrin receptor (hTfR) into phospholipid vesicles using either dialysis, dilution, or gel filtration. Several different detergents and phospholipids and a variety of lipid-to-protein weight ratios were tested. Preformed vesicles as well as detergent-solubilized phospholipids were used. Reconstituted mixtures were fractionated by sucrose density gradient centrifugation and screened for ferritransferrin binding activity, and peak fractions were analyzed by electron microscopy. The efficiency of reconstitution was strongly influenced by the choice of the phospholipids used and the reconstitution method. Best results were obtained when hTfR was dissolved with octylpolyoxyethylene, mixed with detergent-solubilized soy bean lecithin, and reconstituted by slow dialysis. The data show that hTfR capable of binding ferritransferrin was reconstituted into vesicles with an irregular surface and many protrusions. In addition the reconstitution of hTfR resulted in the formation of tubular structures proceeding from the vesicle surface, which may serve as an in vitro model for future studies on the relevance of self-assembly processes for cellular endocytosis.
Fuchs
H
H
Institut für Klinische Chemie und Biochemie, Virchow-Klinikum der Humboldt Universität zu Berlin, Germany.
Gessner
R
R
Tauber
R
R
Ghosh
R
R
D002499:Centrifugation, Density Gradient; D005260:Female; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D008854:Microscopy, Electron; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011990:Receptors, Transferrin / Q000378:metabolism* / Q000648:ultrastructure; D014168:Transferrin / Q000378:metabolism
D008051:Lipid Bilayers; D011990:Receptors, Transferrin; D014168:Transferrin