46] In vitro protein translocation across microsomal membranes of Saccharomyces cerevisiae

Abstract

Publisher Summary This chapter describes the preparation of all components required for co- or posttranslational translocation assays across microsomal membranes of Saccharomyces cerevisiae. Bacteriophage SP6 and T7 RNA polymerases are used to obtain transcripts that contain cap analogs at their 5' ends and are efficiently translated in several cell extracts. This has been possible owing to the ability of both polymerases to utilize an analog diguanosine 5'-Ixiphosphate, of the cap structure found in the 5' end of eukaryotic mRNA, as the first nucleotide during the initiation of RNA synthesis. The coding sequences of several secretory proteins are subcloned in plasmid vectors containing the bacteriophage SP6 RNA polymerase promoter or the T7 RNA polymerase promoter. In most cases, these plasmids produced transcripts active in translation. However, the 5'-untranslated sequences of some genes diminished the translational activity of the mRNAs drastically. This difficulty has been overcame by subcloning these genes into the pSP64T vector, thus replacing the natural γ-untranslated sequences with the 5'-untranslated sequences of Xenopus β-globin mRNA.