Physical determinants of intermembrane protein transfer.
Intermembrane protein transfer between erythrocytes and phospholipid vesicles was examined under a variety of conditions to investigate physical factors governing this process. Human erythrocytes were incubated with sonicated dimyristoylphosphatidylcholine vesicles containing trace [14C]dipalmitoylphosphatidylcholine. Protein-vesicle complexes were separated from cells and from membrane fragments by density gradient centrifugation. The yield of isolated protein vesicles was determined from the 14C-vesicle marker; protein compositions were analyzed by SDS-polyacrylamide gel electrophoresis. Enzymatic removal of portions of the cytoplasmic or exoplasmic domains of cell membrane proteins had little effect on the extent of protein transfer. Membrane additives such as cholate produced a 2-fold increase in protein-vesicle yield. The selectivity of protein transfer from erythrocytes was influenced by the lipid composition of recipient vesicles: inclusion of cholesterol increased band 3 content while the presence of anionic phospholipids reduced transfer. Proteins transferred from 32P-labeled cells differed in specific radioactivity from bulk cell proteins: glycophorin, highly phosphorylated in the cell membrane, showed no detectable labeling in the corresponding protein-vesicle band. These observations suggest that cell-to-vesicle protein transfer is insensitive to bulk steric and electrostatic properties of cell membranes, but enhanced by membrane defects. Recipient membrane composition influences the selectivity of transferred proteins and may reveal subtle differences in the membrane association of protein subpopulations.
Waters
S I
SI
Department of Chemistry, Stanford University, California 94305, USA.
Sen
R
R
Brunauer
L S
LS
Huestis
W H
WH
D019794:2,3-Diphosphoglycerate; D000328:Adult; D001457:Anion Exchange Protein 1, Erythrocyte / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology; D002250:Carbon Radioisotopes; D004134:Dimyristoylphosphatidylcholine / Q000097:blood* / Q000494:pharmacology; D004163:Diphosphoglyceric Acids / Q000097:blood; D004591:Electrophoresis, Polyacrylamide Gel; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006461:Hemolysis; D006801:Humans; D007457:Iodine Radioisotopes; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000097:blood* / Q000302:isolation & purification; D008970:Molecular Weight; D011402:Pronase
D001457:Anion Exchange Protein 1, Erythrocyte; D002250:Carbon Radioisotopes; D004163:Diphosphoglyceric Acids; D007457:Iodine Radioisotopes; D008081:Liposomes; D008565:Membrane Proteins; D019794:2,3-Diphosphoglycerate; D011402:Pronase; D002118:Calcium; D004134:Dimyristoylphosphatidylcholine