Targeting proteins to the lumen of endoplasmic reticulum using N-terminal domains of 11 beta-hydroxysteroid dehydrogenase and the 50-kDa esterase

Previous studies identified two intrinsic endoplasmic reticulum (EB) protei ns, 11 beta-hydroxysteroid dehydrogenase, isozyme 1 (11 beta-HSD) and the 5 0-kDa esterase (E3), sharing some amino acid sequence motifs in their N-ter minal transmembrane (TM) domains. Both are type II membrane proteins with t he C terminus projecting into the lumen of the ER, This finding implied tha t the N-terminal TM domains of 11 beta-HSD and E3 may constitute a lumenal targeting signal (LTS). To investigate this hypothesis we created chimeric fusions using the putative targeting sequences and the reporter gene, Aequo rea victoria green fluorescent protein, Transfected COS cells expressing LT S-green fluorescent protein chimeras were examined by fluorescent microscop y and electron microscopic immunogold labeling The orientation of expressed chimeras was established by immunocytofluorescent staining of selectively permeabilized COS cells. In addition, protease protection assays of membran es in the presence and absence of detergents was used to confirm lumenal or the cytosolic orientation of the constructed chimeras. To investigate the general applicability of the proposed LTS, we fused the N terminus of E3 to the N terminus of the NADH-cytochrome b5 reductase lacking the myristoyl g roup and N-terminal 30-residue membrane anchor. The orientation of the cyto chrome b5 reductase was reversed, from cytosolic to lumenal projection of t he active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed b y a specific array of hydrophobic residues terminating with acidic residues , is sufficient for lumenal targeting of single-pass proteins that are stru cturally and functionally unrelated.