Multi-ubiquitination of a nascent membrane protein produced in a rabbit reticulocyte lysate

During a large-scale in vitro translation analysis of a human full-length c DNA bank, we found many clones producing in vitro translation products show ing ladder bands on a fluorogram with the equidistance of about 9 kDa at th e position larger than the molecular mass expected from the open reading fr ame. We have analyzed a clone showing a typical pattern of the ladder bands . This clone encoded a 188-amino acid polypeptide containing a putative tra nsmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi app aratus. The ladder bands were observed in a rabbit reticulocyte lysate syst em, but not in a wheat germ extract system. Addition of the glutathione S-t ransferase-fused ubiquitin into the lysate caused upward shifts of the ladd er bands. Addition of microsomal membranes prevented the formation of the l adder bands. Time course experiments demonstrated that the in vitro transla tion products increased in the presence of microsomal membranes, but were g radually degraded in their absence. These results suggest that the ladder f ormation resulted from the ubiquitination of misfolded polypeptide that fai led to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate syste m.