A membrane preparation that contains proteins characteristic of the rough endoplasmic reticulum.
We describe a procedure for disassembling rat liver rough microsomes, which allows the purification of the rough endoplasmic reticulum (ER) membrane. Membrane-bound ribosomes and adsorbed proteins are first detached by washing rough microsomes with 5 mM Na-pyrophosphate. In a second step, the vesicle membrane is opened by digitonin, with concomitant release of the luminal content. The purification is monitored at each step by electron microscopy, and by assaying chemical constituents (protein, phospholipid, RNA) and marker enzymes for the main subcellular organelles. The final membrane preparation is representative of the ER, since it contains 24.1% of the liver glucose 6-phosphatase with a relative specific activity of 14.2. Contaminants represent less than 5% of its protein content. SDS-polyacrylamide gel electrophoresis, followed by immunoblot analysis, reveals that the ribophorins I and II, two established markers of the rough (d) domain are still present in the final membrane preparation. It also contains the docking protein (or signal recognition particle receptor) and protein disulfide isomerase, and has conserved the functional capacity to remove co- and post-translationally the signal peptide of pre-secretory proteins. The membrane preparation is suitable for studies on the polypeptide composition of the d domain.
Amar-Costesec
A
A
International Institute of Cellular and Molecular Pathology, Université Catholique de Louvain, Brussels, Belgium.
Hortsch
M
M
Turu
C
C
D000818:Animals; D002458:Cell Fractionation; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000032:analysis* / Q000201:enzymology / Q000648:ultrastructure; D005260:Female; D007425:Intracellular Membranes / Q000032:analysis* / Q000201:enzymology / Q000648:ultrastructure; D008565:Membrane Proteins / Q000032:analysis*; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000032:analysis* / Q000648:ultrastructure; D010455:Peptides / Q000032:analysis; D010743:Phospholipids / Q000032:analysis; D012313:RNA / Q000032:analysis; D051381:Rats; D011919:Rats, Inbred Strains; D012270:Ribosomes / Q000648:ultrastructure; D012697:Serine Endopeptidases
D008565:Membrane Proteins; D010455:Peptides; D010743:Phospholipids; D012313:RNA; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase