Protein translocation in the endoplasmic reticulum. Ultraviolet light induces the noncovalent association of nascent peptides with translocon proteins.
We have photolyzed cell-free translation systems synthesizing beta-lactamase with 254-nm ultraviolet light. In the presence of canine rough microsomes (RM), incomplete chains of beta-lactamase became enriched relative to the full-length molecule in pellet fractions obtained following photolysis and alkaline carbonate extraction. In addition, high molecular weight aggregates were present on SDS-polyacrylamide gels and occurred only when translocation-competent microsomal membranes were used in translation mixtures. The incomplete chains and high molecular weight aggregates were not obtained when RM were inactivated by reaction with N-ethylmaleimide. The incomplete chains did not bind to concanavalin A-Sepharose, indicating that they had not sedimented as a result of being covalently cross-linked to membrane glycoproteins. Both photolysis and alkaline carbonate extraction were required to produce the results. Nascent peptides that were not exposed to alkaline carbonate following photolysis did not appear as high molecular weight bands on SDS-polyacrylamide gels. The high molecular weight aggregates therefore represent denatured protein complexes that contain nascent peptides and microsomal translocon proteins. The results suggest that the translocon is a large proteinaceous complex and that at least a portion of it, when denatured, migrates at a molecular mass of approximately 205 kDa.
Anderson
L
L
Division of Life Sciences, University of Texas, San Antonio 78249-0662.
Denny
J B
JB
D000818:Animals; D002474:Cell-Free System; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D005033:Ethylmaleimide / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism*; D010179:Pancreas / Q000378:metabolism; D010782:Photolysis; D014176:Protein Biosynthesis / Q000187:drug effects / Q000528:radiation effects; D011691:Puromycin / Q000494:pharmacology; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D014466:Ultraviolet Rays; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D006023:Glycoproteins; D008562:Membrane Glycoproteins; D011691:Puromycin; D003513:Cycloheximide; D001618:beta-Lactamases; D005033:Ethylmaleimide