Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity.
We have used the membrane-impermeable, thiol-cleavable, crosslinker 3,3'-dithio bis (sulfosuccinimidylpropionate) to identify proteins that are in the vicinity of membrane-bound ribosomes of the RER. A specific subset of RER proteins was reproducibly crosslinked to the ribosome. Immunoblot analysis of the crosslinked products with antibodies raised against signal recognition particle receptor, ribophorin I, and the 35-kD subunit of the signal sequence receptor demonstrated that these translocation components had been crosslinked to the ribosome, but each to a different extent. The most prominent polypeptide among the crosslinked products was a 180-kD protein that has recently been proposed to be a ribosome receptor (Savitz, A.J., and D.I. Meyer, 1990. Nature (Lond.). 346: 540-544). RER membrane proteins were reconstituted into liposomes and assayed with radiolabeled ribosomes to determine whether ribosome binding activity could be ascribed to the 180-kD protein. Differential detergent extraction was used to prepare soluble extracts of microsomal membrane vesicles that either contained or lacked the 180-kD protein. Liposomes reconstituted from both extracts bound ribosomes with essentially identical affinity. Additional fractionation experiments demonstrated that the bulk of the ribosome binding activity present in detergent extracts of microsomal membranes could be readily resolved from the 180-kD protein by size exclusion chromatography. Taken together, we conclude that the 180-kD protein is in the vicinity of membrane bound ribosomes, yet does not correspond to the ribosome receptor.
Collins
P G
PG
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Gilmore
R
R
D000818:Animals; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015151:Immunoblotting; D007425:Intracellular Membranes / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D011485:Protein Binding; D012270:Ribosomes / Q000378:metabolism*; D013388:Succinimides
D003432:Cross-Linking Reagents; D008081:Liposomes; D008565:Membrane Proteins; D013388:Succinimides; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate)