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J Biol Chem, Vol. 274, Issue 20, 14122-14129, May 14, 1999

Targeting Proteins to the Lumen of Endoplasmic Reticulum Using N-terminal Domains of 11-Hydroxysteroid Dehydrogenase and the 50-kDa Esterase

Hassan Mziaut, George Korza, Arthur R. Hand^§, Craig Gerard^¶, and Juris Ozols

From the  Department of Biochemistry and ^§ Electron Microscopy, University of Connecticut Health Center, Farmington, Connecticut 06030-3305 and the ^¶ Department of Pediatrics, Children's Hospital, Harvard Medical School, Boston, Massachusetts 02115

Previous studies identified two intrinsic endoplasmic reticulum (ER) proteins, 11-hydroxysteroid dehydrogenase, isozyme 1 (11-HSD) and the 50-kDa esterase (E3), sharing some amino acid sequence motifs in their N-terminal transmembrane (TM) domains. Both are type II membrane proteins with the C terminus projecting into the lumen of the ER. This finding implied that the N-terminal TM domains of 11-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, Aequorea victoria green fluorescent protein. Transfected COS cells expressing LTS-green fluorescent protein chimeras were examined by fluorescent microscopy 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 membranes 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 group and N-terminal 30-residue membrane anchor. The orientation of the cytochrome b5 reductase was reversed, from cytosolic to lumenal projection of the active domain. These observations establish that an amino acid sequence consisting of short basic or neutral residues at the N terminus, followed by a specific array of hydrophobic residues terminating with acidic residues, is sufficient for lumenal targeting of single-pass proteins that are structurally and functionally unrelated.

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