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

Abstract

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.

Hexose-6-phosphate dehydrogenase: linking endocrinology and metabolism in the endoplasmic reticulum

“…HSD11B1 enzyme is an integral membrane protein of the ER with luminal active site (Ozols 1995, Mziaut et al 1999, Odermatt et al 1999. It catalyzes the interconversion between cortisone and cortisol using NADPH or NADP C coenzyme.…”

Mutations of key hydrophobic surface residues of 11β‐hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system

“…14 11b-HSD1 is a member of the short-chain dehydrogenase reductase (SDR) superfamily of enzymes. Unusually for an SDR enzyme, 11b-HSD1 has an N-terminal membrane anchor, 15,16 with a small portion of the N terminal segment of the enzyme present in the cytosol.…”

11β-Hydroxysteroid dehydrogenase type 1 shRNA ameliorates glucocorticoid-induced insulin resistance and lipolysis in mouse abdominal adipose tissue

“…However, tissuespecific GC action not only depends on circulating GCs levels but can also be regulated by an intracellular endoplasmic reticulum (ER) lumen resident enzyme, 11␤-hydroxysteroid dehydrogenase type 1 (11␤-HSD1), that converts inactive cortisone (11-dehydrocorticosterone in rodents) to active cortisol (corticosterone) and therefore amplifies intracellular GC action, particularly in adipose tissue (6,21,24,41). 11␤-HSD1 is regulated by its cofactor NADPH, which is provided by the enzyme hexose-6-phosphate dehydrogenase (H6PDH) (32,34). In the ER lumen, H6PDH can metabolize glucose 6-phosphate and NADP to generate NADPH, thus ensuring 11␤-HSD1 activity.…”