Abstract
Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within transfected mouse hepatoma cells and is subjected to intracellular degradation (Le, A., Graham, K., and Sifers, R.N. (1990) J. Biol. Chem. 265, 14001-14007). The retained protein undergoes a discrete size reduction that results from the modification of its endoglycosidase H-sensitive oligosaccharides and is inhibited by 1-deoxymannojirimycin. Metabolic poisons and inhibitors of protein synthesis perturb the intracellular degradation of the retained protein but do not affect its size reduction. The ability of metabolic poisons to influence the degradation of the PiZ variant in cells treated with brefeldin A indicates that export of the macromolecule from the endoplasmic reticulum (ER) is not the energy-dependent component of its degradation. Subcellular fractionation experiments have verified that both the size reduction and degradation of the retained PiZ variant occur within the rough ER. Finally, sedimentation velocity centrifugation analysis of radiolabeled cell extracts has indicated that approximately 80% of the PiZ variant consists as soluble aggregates immediately after its synthesis. An inability to detect more extensive aggregation during the retention period supports our previous conclusion that only a small fraction of the macromolecules actually form large insoluble aggregates (Graham, K.S., Le, A., and Sifers, R.N. (1990) J. Biol. Chem. 265, 20463-20468). Overall, these findings indicate that soluble aggregates of the PiZ variant are degraded within the ER by a mechanism sensitive to inhibitors of protein synthesis.
Highlights
Soluble Aggregates of the Human P i Z crl-Antitrypsin Variant Are Degraded within theEndoplasmic Reticulumby a Mechanism Sensitive toInhibitors of Protein Synthesis*
T o determine of the radiolabeled macromolecules appeared in themedia of whether the oligosaccharide-dependent size reduction of the these cells within 1h following their synthesis and exhibited retainedPiZvariantis caused by adelayedhydrolysis of a mass of 56 kDa (Fig. lA)which results from the additionof glucose residues, HlA/RSVATZ-8 cells were preincubated charged sialicacidresidues to the oligosaccharidemoieties and thenradiolabeled in media containing 1-deoxynorjirimywithinthetrans Golgi networkpriortosecretion [25]
C, dishes of HlA/M15 cells were pulseradiolabeled for 5 min with 300 pCi of TRAN"S Label and chased for 2.5 h in chase media a t 15 "C with (+) or without (-) 1mM of 1deoxymannojirimycin.Human AAT was immunoisolated from cell extracts and immunocomplexes were fractionated by SDSPAGE
Summary
Was identical to thaot f the pulse-radiolabeled protein in SDSPAGE (Fig. 1B). These findings indicated that the size re-. T o determine of the radiolabeled macromolecules appeared in themedia of whether the oligosaccharide-dependent size reduction of the these cells within 1h following their synthesis and exhibited retainedPiZvariantis caused by adelayedhydrolysis of a mass of 56 kDa (Fig. lA)which results from the additionof glucose residues, HlA/RSVATZ-8 cells were preincubated charged sialicacidresidues to the oligosaccharidemoieties and thenradiolabeled in media containing 1-deoxynorjirimywithinthetrans Golgi networkpriortosecretion [25]. In cin (dNJ) tospecifically inhibit the actionof ER a-glucosidcontrast, only 15% of the pulse-radiolabeled PiZ variant was ases I and 11 This manipulation retarded themobility of the secretedfromtransfected mouse hepatoma cells (cell line immunoisolatedPiZvariantinSDS-PAGE (Fig. 2 A ) and. Althoughthe retained pro- compartments contain a-mannosidases that are inhibitbeyd themannose analogue1-deoxymannojirimycin (dMM) [27, 28], we tested whether thiscompound would hinder theoligosaccharide-dependent size reduction of the retained PiZ variant.
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