Determination of lumenal orientation of microsomal 50-kDa esterase/N-deacetylase.

Abstract

The amino acid arrangements responsible for the insertion and specific lumenal orientation of proteins having an uncleaved signal-peptide-like anchor are poorly understood. A 50-kDa protein having a hydrophobic N-terminus similar to the lumenal glycoprotein 11beta-hydroxysteroid dehydrogenase [Ozols, J. (1995) J. Biol. Chem. 270, 2305-2312] was identified in detergent-solubilized microsomes. The posttranslational modifications and the membrane orientation of the 50-kDa protein were investigated using the approaches of protein structure analysis. Sequence analysis of the entire 50-kDa protein showed a lack of structural relatedness to the steroid dehydrogenase beyond the membrane binding segment. Structure analysis of peptides revealed that carbohydrate is attached at Asn-77 and Asn-281, implying that these sites of the 50-kDa protein are oriented toward the lumenal side of the endoplasmic membrane (ER). Specific enzymatic deglycosylation on the intact protein identified the two glycans as high mannose carbohydrate rather than of the complex type, suggesting that the protein had not undergone further trafficking steps beyond the lumen of ER. Chemical modification of cysteinyl residues showed a lack of free thiols in the intact protein. Peptide mapping identified one disulfide bond between Cys-115 and -340 further restricting the bulk of the protein to the lumenal compartment. Proteolysis of intact and solubilized microsomes showed that the 50-kDa protein is resistant to fragmentation at the conditions which led to the removal of the membranous segments from cytochrome b5 and the NADH-cytochrome b5 reductase. The proposed model of the 50-kDa protein predicts one transmembrane segment at the N-terminus, flanked by positively charged residues on the cytosolic surface and negatively charged residues on the lumenal side of the hydrophobic domain, with most of the polypeptide projecting into the lumen of the ER. The stated similarities in the topology between 11beta-steroid dehydrogenase and 50-kDa protein envision their transmembrane segment consisting of a basic residue(s) followed by an array of some 17 hydrophobic residues containing the Ala-Tyr-Tyr-X-Tyr cluster, where X represents a hydrophobic amino acid, which terminates with acidic residues. It is proposed here that such a motif may constitute a lumenal targeting signal for a set of single-membrane-spanning proteins that are otherwise structurally and functionally unrelated.