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.
Skip Nav Destination
Article navigation
15 August 1991
Article|
August 15 1991
Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity.
P G Collins,
P G Collins
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Search for other works by this author on:
R Gilmore
R Gilmore
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Search for other works by this author on:
P G Collins
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
R Gilmore
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Online ISSN: 1540-8140
Print ISSN: 0021-9525
J Cell Biol (1991) 114 (4): 639–649.
Citation
P G Collins, R Gilmore; Ribosome binding to the endoplasmic reticulum: a 180-kD protein identified by crosslinking to membrane-bound ribosomes is not required for ribosome binding activity.. J Cell Biol 15 August 1991; 114 (4): 639–649. doi: https://doi.org/10.1083/jcb.114.4.639
Download citation file:
Sign in
Don't already have an account? Register
Client Account
You could not be signed in. Please check your email address / username and password and try again.
Could not validate captcha. Please try again.
Sign in via your Institution
Sign in via your InstitutionSuggested Content
Email alerts
Advertisement
Advertisement