Reconstitution of translocation-competent membrane vesicles from detergent-solubilized dog pancreas rough microsomes.
Dog pancreas rough microsomes were solubilized in 1% octyl beta-glucoside, and membrane vesicles were reconstituted by slow 30-fold dilution with a buffer of low ionic strength. Asymmetric assembly of the membranes occurred during reconstitution since the vesicles formed contained ribosomes bound only to the vesicular outer surfaces. The reconstituted vesicles were similar in protein composition to native rough microsomes, although these vesicles were largely devoid of luminal-content proteins. These reconstituted vesicles could translocate and process nascent secretory (human placental lactogen) and membrane proteins (influenza hemagglutinin and rat liver ribophorin I) synthesized in cell-free translation systems programmed with the corresponding mRNAs. Signal cleavage and N-glycosylation only occurred when the reconstituted membranes were present during translation, providing evidence that the translocation apparatus was asymmetrically assembled into the reconstituted membranes. When a supernatant lacking ribosomes and particles greater than 50S from centrifuging the detergent-solubilized microsomes at high speed was used for reconstitution, smooth-surfaced membrane vesicles were obtained that, except for the absence of ribosomal proteins, were similar in protein composition to that of the reconstituted vesicles from total solubilized rough microsomes. The reconstituted smooth-surfaced vesicles, however, were totally inactive in cotranslational processing and translocation of nascent polypeptides. These findings suggest that ribosomes and/or large macromolecular complexes, not dissociated under our solubilization conditions, are essential for in vitro assembly of a functional translocation apparatus.
Yu
Y H
YH
Department of Cell Biology, New York University Medical Center, NY 10016.
Zhang
Y Y
YY
Sabatini
D D
DD
Kreibich
G
G
D000818:Animals; D001692:Biological Transport; D002458:Cell Fractionation; D003902:Detergents; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D005960:Glucosides; D006031:Glycosylation; D006388:Hemagglutinins; D007425:Intracellular Membranes / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D008854:Microscopy, Electron; D008861:Microsomes / Q000378:metabolism / Q000648:ultrastructure*; D008970:Molecular Weight; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure*
D003902:Detergents; D005960:Glucosides; D006388:Hemagglutinins; D008565:Membrane Proteins; C018619:octyl-beta-D-glucoside