A large presecretory protein translocates both cotranslationally, using signal recognition particle and ribosome, and post-translationally, without these ribonucleoparticles, when synthesized in the presence of mammalian microsomes.
Translocation of large presecretory proteins into the mammalian endoplasmic reticulum requires the ribonucleoparticles, signal recognition particle, and ribosome and is tightly coupled to ongoing protein synthesis. We have shown previously that small presecretory proteins can translocate post-translationally in a reaction that does not require these ribonucleoparticles. We now report that one large protein, a synthetic hybrid between preprocecropin A and dihydrofolate reductase, translocates both cotranslationally (with the aid of signal recognition particle and ribosome) and post-translationally (without the involvement of these ribonucleoparticles) during its in vitro synthesis in the presence of dog pancreas microsomes. The distinction between these two modes of translocation was made possible by adding methotrexate to the translocation reaction. Methotrexate can only form a tight complex with those preprocecropin A-dihydrofolate reductase hybrid chains that have completed their synthesis and folded, but in forming this tight complex, this drug prevents translocation of the dihydrofolate reductase domain across the membrane.
Schlenstedt
G
G
Institut für Physiologische Chemie der Universität München, Federal Republic of Germany.
Gudmundsson
G H
GH
Boman
H G
HG
Zimmermann
R
R
D000595:Amino Acid Sequence; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D005493:Folic Acid Antagonists / Q000494:pharmacology; D007301:Insect Hormones / Q000235:genetics*; D019476:Insect Proteins; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D008861:Microsomes / Q000187:drug effects / Q000378:metabolism; D008969:Molecular Sequence Data; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000235:genetics*; D012261:Ribonucleoproteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D014158:Transcription, Genetic; D014357:Trypsin / Q000494:pharmacology
C059037:CecA2 protein, Bombyx mori; D005493:Folic Acid Antagonists; D007301:Insect Hormones; D019476:Insect Proteins; D011498:Protein Precursors; D021382:Protein Sorting Signals; D012261:Ribonucleoproteins; D003513:Cycloheximide; D013762:Tetrahydrofolate Dehydrogenase; D014357:Trypsin; D008727:Methotrexate