Uncoupling of translocation across microsomal membranes from biosynthesis of influenza virus hemagglutinin.

This communication presents our recent studies on the biosynthesis of influenza virus hemagglutinin (HA) in a mammalian-cell-free system and its translocation across microsomal membranes. RNAs coding for wild-type (full-length) and mutant (truncated) forms of HA were generated by in vitro transcription by using bacteriophage T7 DNA-dependent RNA polymerase. These RNAs were translated in a rabbit reticulocyte system that was supplemented with dog pancreas membranes, either before translation was initiated or after it had been artificially terminated with the antibiotic cycloheximide. All forms of HA could be cotranslationally translocated. However, only truncated molecules (83% of full length) could translocate after protein synthesis had been terminated. Posttranslational translocation was dependent on the presence of a functional N-terminal signal sequence and occurred only in the presence of ribosomes. The molecular mechanism of protein targeting and translocation across the membrane of the endoplasmic reticulum is discussed based on the signal hypothesis.