Lumenal location of the microsomal beta-glucuronidase-egasyn complex.

Abstract

Abstract. Mouse liver l~-glucuronidase is stabilized within microsomal vesicles by complexation with the accessory protein egasyn. The location of the 13-glucuronidase-egasyn complex and free egasyn within microsomal vesicles was investigated. Surprisingly, it was found that neither the complex nor free egasyn are intrinsic membrane components. Rather, both are either free within the vesicle lumen or only weakly bound to the inside of the vesicle membrane. This conclusion was derived from release studies using low concentrations of Triton X-100 or controlled sonication. Both the intact complex and free egasyn were released in parallel with lumenal proteins, not with intrinsic membrane components. Also, 13-glucuronidase was protected from digestion by proteinase K by the membrane of microsomal vesicles. The hydrophilic nature of both the complex and free egasyn was confirmed by phase separation experiments with the detergent Triton X-114. Egasyn is one of an unusual group of esterases that, despite being located within the lumen or only weakly bound to the lumenal surface of the endoplasmic reticulum, do not enter the secretory pathway. ~-Glucuronidase is an unusual acid hydrolase in that large amounts (20-40%) of stable enzyme exist in the microsomal comparmaent of liver in addition to the usual lysosomal location. A wide variety of biochemical and genetic information indicates that the same polypeptide is found at both subcellular sites and that complex formation with the accessory protein, egasyn, is required for stabilization of ~glucuronidase in microsomes (reviewed in 24). The system thus serves as a model for the specific subceUular localization of enzymes. Mutant mice that lack egasyn have no stable microsomal I~-glucuronidase (14). More recent studies have shown that egasyn is associated exclusively with the precursor form of 13-glucuronidase in microsomes (9) and that egasyn is identical with mouse esterase-22 (26,27).It has been postulated that egasyn stabilizes the l~-glucuronidase complex in microsomes by serving as an integral membrane anchor peptide (24). Others (12) have suggested that microsomal I~-glucuronidase serves in a membrane structural role. In this paper we present evidence that the microsomal 13-glucuronidase-egasyn complex, rather than being an integral membrane component, surprisingly is either free within the lumen of microsomal vesicles or very weakly attached to the inside of these vesicles. These results suggest, in turn, that the binding protein, egasyn, stabilizes 13-glucuronidase within the microsomal lumen. Materials and Methods AnimalsC57BL/6J, Mus musculus molossinus and Mus spretus mice were raised in the animal facilities of Roswell Park Memorial Institute. Mice 2-4 mo old and of both sexes were used. Unless otherwise indicated, C57BL/6J mice were used. Radiolabeling ExperimentsRadiolabeling of membrane and secretory components of microsomes was done according to the method of Kreibich and Sabatini (20). Membrane phospholipids were labeled by injecting mice intraperitoneal...

A Secreted Tyrosine Kinase Acts in the Extracellular Environment

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Cell type-specific post-Golgi apparatus localization of a "resident" endoplasmic reticulum glycoprotein, glucosidase II.

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