The COOH terminus of several liver carboxylesterases targets these enzymes to the lumen of the endoplasmic reticulum.
To investigate the potential role of the COOH-terminal peptides in retaining a family of soluble carboxylesterases in the lumen of the endoplasmic reticulum, the pI 6.1 esterase cDNA has been cloned into the pKCR3 vector for transient expression in COS cells. The plasmid-encoded product appeared to be identical to the authentic enzyme: it was active on alpha-naphthyl acetate and behaved as a homotrimer of noncovalently bound 60-kDa subunits which contain a single, endo-beta-N-acetylglucosaminidase H-sensitive oligosaccharide chain. This enzyme was retained in the transgenic COS cells. In contrast, a mutated form ending in HVER-COOH was secreted, indicating that the natural terminus HVEL-COOH contains topogenic information, with the ultimate Leu residue as an essential part. Variants of pI 6.1 esterase ending in HIEL-COOH, or HTEL-COOH were retained in cells to the same extent as the wild-type protein. Therefore, the sequences HIEL and HTEL present at the COOH termini of several liver esterases (rabbit forms 1 and 2, human esterase, mouse egasyn, and rat pI 6.4 esterase) most likely have a function in their localization in the endoplasmic reticulum. Finally, an HDEL-COOH variant of pI 6.1 esterase was also normally retained, demonstrating that this signal can be correctly decoded by the sorting machinery of mammalian cells. Cell retention signals of the type HXEL-COOH appear to be common in higher eukaryotes and tolerate considerable variation at the antepenultimate X residue.
Robbi
M
M
Laboratoire de Chimie Physiologique, Université de Louvain, Brussels, Belgium.
Beaufay
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006596:Hexosaminidases; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010957:Plasmids; D011817:Rabbits; D051381:Rats
D004247:DNA; D002265:Carboxylic Ester Hydrolases; D006596:Hexosaminidases