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• W1969809368 abstract "In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded α1-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861–5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed. In the early secretory pathway, a distinct set of processing enzymes and family of lectins facilitate the folding and quality control of newly synthesized glycoproteins. In this regard, we recently identified a mechanism in which processing by endoplasmic reticulum mannosidase I, which attenuates the removal of glucose from asparagine-linked oligosaccharides, sorts terminally misfolded α1-antitrypsin for proteasome-mediated degradation in response to its abrogated physical dissociation from calnexin (Liu, Y., Choudhury, P., Cabral, C., and Sifers, R. N. (1999) J. Biol. Chem. 274, 5861–5867). In the present study, we examined the quality control of genetic variant PI Z, which undergoes inappropriate polymerization following biosynthesis. Here we show that in stably transfected hepatoma cells the additional processing of asparagine-linked oligosaccharides by endoplasmic reticulum mannosidase II partitions variant PI Z away from the conventional disposal mechanism in response to an arrested posttranslational interaction with calnexin. Intracellular disposal is accomplished by a nonproteasomal system that functions independently of cytosolic components but is sensitive to tyrosine phosphatase inhibition. The functional role of ER mannosidase II in glycoprotein quality control is discussed. endoplasmic reticulum α1-antitrypsin polyacrylamide gel electrophoresis UDP-glucose:glycoprotein glucosyltransferase In the endoplasmic reticulum (ER),1 an assortment of molecular chaperones and folding enzymes facilitate the conformational maturation of newly synthesized polypeptides destined for deployment to the cell surface as biologically active proteins (1Gething M.J. Sambrook J. Nature. 1992; 355: 33-45Crossref PubMed Scopus (3590) Google Scholar). In recent years, a picture has emerged that describes how asparagine-linked glycosylation, in combination with several independently acting enzymes, facilitates glycoprotein folding (for reviews, see Refs. 2Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (559) Google Scholar and3Hammond E. Helenius A. Curr. Opin. Cell Biol. 1995; 7: 523-529Crossref PubMed Scopus (587) Google Scholar). In a widely accepted model (4Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (712) Google Scholar), the partial deglucosylation of asparagine-linked Glc3Man9GlcNAc2 induces cotranslational physical interaction between glycoproteins and members of a small family of lectins, each of which recognizes the monoglucosylated glycan as ligand (2Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (559) Google Scholar). Dissociation of the complex coincides with the removal of glucose by glucosidase II (5Hebert D.N. Foellmer B. Helenius A. Cell. 1995; 81: 425-433Abstract Full Text PDF PubMed Scopus (488) Google Scholar). In the absence of conformational maturation, UDP-glucose:glycoprotein glucosyltransferase (UGTR) functions as a folding sensor (6Parodi A.J. Mendelzon D.H. Lederkremer G.Z. J. Biol. Chem. 1983; 258: 8260-8265Abstract Full Text PDF PubMed Google Scholar) that recognizes structural determinants common to nonnative glycoprotein structure (7Sousa M. Parodi A.J. EMBO J. 1995; 14: 4196-4203Crossref PubMed Scopus (241) Google Scholar, 8Fernandez F. D'Alessio C. Fanchiotti S. Parodi A.J. EMBO J. 1998; 17: 5877-5886Crossref PubMed Scopus (41) Google Scholar). Reglucosylation of asparagine-linked oligosaccharides induces the reassembly of folding intermediates with calnexin (4Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (712) Google Scholar). As such, reversible glucosylation hinders premature exit from the ER (2Helenius A. Mol. Biol. Cell. 1994; 5: 253-265Crossref PubMed Scopus (559) Google Scholar,4Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (712) Google Scholar, 9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) until correctly folded molecules that are no longer substrates for UGTR are released from the lectin-mediated retention cycle (4Hammond C. Braakman I. Helenius A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 913-917Crossref PubMed Scopus (712) Google Scholar). As a rule, failure to attain conformational maturation following biosynthesis results in the selective elimination of misfolded polypeptides and unassembled protein complexes by a relatively stringent mechanism of conformation-based quality control (10Klausner R.D. Sitia R. Cell. 1990; 62: 611-614Abstract Full Text PDF PubMed Scopus (455) Google Scholar, 11Ellgard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1887Crossref PubMed Scopus (1062) Google Scholar). Molecular characterization of primary and secondary disposal systems, plus the identification of the full repertoire of quality control machinery, is currently under intense investigation (for a review, see Ref. 11Ellgard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1887Crossref PubMed Scopus (1062) Google Scholar). Because the efficiency of modification by UGTR and glucosidase II is sensitive to the number of mannose units within the asparagine-linked oligosaccharide (6Parodi A.J. Mendelzon D.H. Lederkremer G.Z. J. Biol. Chem. 1983; 258: 8260-8265Abstract Full Text PDF PubMed Google Scholar, 13Grinna L.S. Robbins P.W. J. Biol. Chem. 1980; 255: 2255-2258Abstract Full Text PDF PubMed Google Scholar), it is possible that oligosaccharide processing represents the meeting point between protein folding and quality control pathways. For this reason, recent work has focused toward elucidating the potential role of processing mannosidases in glycoprotein quality control (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 14Yang M. Omura S. Bonifacino J.S. Weissman A.M. J. Exp. Med. 1998; 187: 835-846Crossref PubMed Scopus (201) Google Scholar, 15Jakob C.A. Burda P. Roth J. Aebi M J. Cell Biol. 1998; 152: 1223-1233Crossref Scopus (301) Google Scholar). In addition to its fundamental importance in normal cell physiology (16Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1892Crossref PubMed Scopus (910) Google Scholar), the process of quality control in the early secretory pathway has been implicated as a key factor in the molecular pathogenesis associated with several human disorders (17Thomas P.J. Qu B.-H. Pedersen P.L. Trends Biochem. Sci. 1995; 20: 456-459Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 18Choudhury P. Liu Y. Sifers R.N. News Physiol. Sci. 1997; 12: 162-165Google Scholar). To this end, a major physiologic role for the monomeric secretory glycoprotein α1-antitrypsin (AAT) is to prevent the destruction of lung elastin. The hindered secretion and disposal of allelic variants from liver hepatocytes, the predominant site of biosynthesis (19Carlson J.A. Rogers B.B. Sifers R.N. Hawkins H.K. Finegold M.J. Woo S.L.C. J. Clin. Invest. 1988; 82: 26-36Crossref PubMed Scopus (120) Google Scholar), can lead to plasma AAT deficiency. A severe deficiency of the plasma protein is known to function as a heritable risk factor for the development of chronic obstructive lung disease (for reviews, see Refs.20Sifers R.N. Shen R.-F. Woo S.L.C. Mol. Biol. Med. 1989; 6: 127-135PubMed Google Scholar and 21Sifers R.N. Finegold M.J. Woo S.L.C. Semin. Liver Dis. 1992; 12: 301-310Crossref PubMed Scopus (42) Google Scholar). Gene expression studies performed in stably transfected murine hepatoma cells have allowed for the characterization of AAT quality control mechanisms in a physiologically relevant model system (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 22Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1989; 263: 7330-7335Abstract Full Text PDF Google Scholar, 23Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar, 24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar, 25Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). The truncation of carboxyl-terminal amino acids in genetic variant PI QO Hong Kong (null(Hong Kong)) (22Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1989; 263: 7330-7335Abstract Full Text PDF Google Scholar) precludes conformational maturation following biosynthesis, resulting in its lectin-mediated intracellular retention prior to disposal (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). Recently, we (26Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar) proposed a model of quality control in which the removal of a single terminal α1,2-linked mannose unit from multiple asparagine-linked oligosaccharides abrogates the physical dissociation of null(Hong Kong) from the ER lectin calnexin (27Ou W.J. Cameron P.H. Thomas D.Y. Bergeron J.J. Nature. 1993; 364: 771-776Crossref PubMed Scopus (486) Google Scholar), leading to its selective degradation by the cytosolic proteasome (28Rivett J.A. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (381) Google Scholar). In this process of “molecular capture,” the attenuated removal of glucose from asparagine-linked oligosaccharides functions as the underlying mechanism by which the misfolded glycoprotein is selected for degradation. In the present study, stably transfected hepatoma cells were used for the molecular characterization of the conformation-based quality control of variant PI Z, the most common severe deficiency variant of human AAT (29Brantly M. Nukiwa Y. Crystal R.G. Am. J. Med. 1989; 84: 13-31Abstract Full Text PDF Google Scholar). A single amino acid substitution at the base of the reactive center loop (12McCracken A.A. Karpichev I.V. Ernaga J.E. Werner E.D. Dillin A.G. Courchesne W.E. Genetics. 1996; 144: 1355-1362Crossref PubMed Google Scholar, 30Stein P.E. Carrell R.W. Nat. Struct. Biol. 1995; 2: 96-113Crossref PubMed Scopus (392) Google Scholar) favors inappropriate polymerization (31Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar) of a late folding intermediate (32Yu M.-H. Lee K.N. Kim J. Nat. Struct. Biol. 1995; 2: 363-367Crossref PubMed Scopus (140) Google Scholar), hindering its secretion from liver hepatocytes. The structural anomaly has been detected by velocity sedimentation (24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar), fluorescence-based and ultrastructural analyses (31Lomas D.A. Evans D.L. Finch J.T. Carrell R.W. Nature. 1992; 357: 605-607Crossref PubMed Scopus (893) Google Scholar), and transverse urea gradient gels following in vitrorefolding (32Yu M.-H. Lee K.N. Kim J. Nat. Struct. Biol. 1995; 2: 363-367Crossref PubMed Scopus (140) Google Scholar). Enhanced secretion of recombinant PI Z bearing site-directed mutations predicted to impede loop-sheet polymerization (33Sidhar S.K. Lomas D.A. Carrell R.W. Foreman R.C. J. Biol. Chem. 1996; 270: 8393-8396Abstract Full Text Full Text PDF Scopus (63) Google Scholar) has confirmed that the structural anomaly plays a predominant role in the intracellular transport defect. The intracellular accumulation of an insoluble fraction of PI Z polymers is responsible, in part, for a heritable form of liver cirrhosis (21Sifers R.N. Finegold M.J. Woo S.L.C. Semin. Liver Dis. 1992; 12: 301-310Crossref PubMed Scopus (42) Google Scholar), and a hindered rate of disposal has been linked to this phenotype (34Wu Y. Whitman I. Molmenti E. Moore K. Hippenmyer P. Perlmutter D.H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9014-9018Crossref PubMed Scopus (229) Google Scholar). Thus, the molecular characterization of PI Z quality control may lead to the development of therapeutic interventions to alleviate the severity of the lung and liver disease. Here we demonstrate that the selective elimination of variant PI Z is accomplished by a nonproteasomal mechanism that is cytosol-independent but sensitive to general inhibitors of tyrosine phosphatase activity. The results of glycosidase inhibitor studies plus coimmunoprecipitation analyses indicate that the combined processing of asparagine-linked oligosaccharides by ER mannosidases I and II (35Moremen K.W. Trimble R.B. Hersovics A. Glycobiology. 1994; 4: 113-125Crossref PubMed Scopus (323) Google Scholar) partitions variant PI Z away from the conventional proteasome-mediated disposal mechanism by preventing posttranslational assembly with calnexin. These data implicate ER mannosidase II as a component of glycoprotein quality control and identify a natural molecular strategy to partition variant PI Z away from the conventional proteasome-mediated disposal pathway. Glycosidase inhibitors were purchased from Toronto Research Chemicals, Inc. and Roche Molecular Biochemicals. All phosphatase inhibitors and proteasome inhibitors, except for lactacystin, were purchased from Calbiochem. Lactacystin was purchased from the E. J. Corey laboratory (Harvard Medical School). All routine buffers and salts were procured from Sigma. Previously established mouse hepatoma cells stably transfected with human genomic DNA encoding human AAT variants (cell lines H1A/M-15, H1A/N13, and H1A/Z8) were used in these analyses (22Sifers R.N. Brashears-Macatee S. Kidd V.J. Muensch H. Woo S.L.C. J. Biol. Chem. 1989; 263: 7330-7335Abstract Full Text PDF Google Scholar, 23Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar, 24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar). Stably transfected clones that exhibited the highest rate of AAT biosynthesis relative to endogenous murine albumin were chosen to mimic the authentic in vivo conditions of hepatocytes. Monolayers of semiconfluent cells were pulse-radiolabeled for 15 min at 37 °C in methionine-free medium (ICN Pharmaceuticals, Inc.) containing [35S]methionine (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) (NEN Life Science Products) and then chased in methionine-free medium for up to 3 h. Cells were lysed with buffered Nonidet P-40 detergent (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar) either immediately following the pulse or at the specified time point during the chase. Experiments involving kifunensine, swainsonine, 1-deoxymannojirimycin, castanospermine, and lactacystin included a 1-h preincubation period prior to pulse radiolabeling. Unless stated otherwise, all other inhibitors were added to cells at the onset of the chase period. Pervanadate was generated from sodium orthovanadate prior to use, as described previously (36Miller S.C. Furniss M.J. J. Biol. Chem. 1998; 273: 32618-32626Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar). Steady-state radiolabeling of cells with [35S]methionine was performed as described previously (37Graham K.S., Le Anhquyen Sifers R.N. J. Biol. Chem. 1990; 265: 20463-20468Abstract Full Text PDF PubMed Google Scholar). Proteins were immunoprecipitated by a 2-h incubation at 4 °C with an excess of specific polyclonal antiserum against human AAT (ICN Pharmaceuticals, Inc.) or murine albumin (Bethyl Laboratories) immobilized to protein G-agarose (Calbiochem) (25Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). Radiolabeled proteins were resolved by SDS-PAGE and detected by fluorographic enhancement of vacuum-dried gels. Quantitation of radiolabel was performed by scintillation counting of excised gel slices (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 25Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). For the detection of insoluble radiolabeled PI Z in pulse-chase experiments, the insoluble pellet resulting from the centrifugation (10,000 × g, 10 min) of the Nonidet P-40 cell lysate was agitated with 1% lithium dodecylsulfate for 10 min. (37Graham K.S., Le Anhquyen Sifers R.N. J. Biol. Chem. 1990; 265: 20463-20468Abstract Full Text PDF PubMed Google Scholar), prior to immunoprecipitation of the released protein and detection by fluorography. Selective permeabilization of the plasma membrane was performed at isotonic conditions as described for human hepatoma cells (38Adeli K. J. Biol. Chem. 1994; 269: 9166-9175Abstract Full Text PDF PubMed Google Scholar). Briefly, confluent monolayers of pulse-radiolabeled cells in semiconfluent 100-mm diameter dishes were washed at 25 °C with CSK buffer (0.3m sucrose, 0.1 m KCl, 2.5 mmMgCl2, 1 mm EDTA, 10 mm PIPES, pH 6.8) and then incubated for 5 min at that same temperature with 3 ml of CSK buffer containing 0.050 mg/ml digitonin (Roche Molecular Biochemicals). The optimal concentration of digitonin needed to efficiently permeabilize >98% of cells was determined by Trypan blue exclusion (39Choudhury P. Liu Y. Bick R.J. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and by the loss of the immunoreactive 20 S proteasome α-subunit. Digitonized cells were washed three times at 25 °C with CSK buffer prior to the initiation of a 3-h incubation at 37 °C in CSK buffer. Following each experiment, AAT was immunoprecipitated from the incubation buffer and the Nonidet P-40 lysate derived from the permeabilized cells, as described for intact cells. Aliquots of the buffered Nonidet P-40 cell lysate or media were subjected to velocity sedimentation in linear 5–20% sucrose gradients in a manner that was identical to that described previously (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar, 25Le A. Steiner J.L. Ferrell G.A. Shaker J.C. Sifers R.N. J. Biol. Chem. 1994; 269: 7514-7519Abstract Full Text PDF PubMed Google Scholar). Variant PI Z was immunoprecipitated from individual gradient fractions, as described above, and then resolved by SDS-PAGE, detected by fluorography, and quantitated by scintillation counting of excised gel pieces. After the electrophoretic transfer of protein from SDS-PAGE gels onto Hybond ECL nitrocellulose membranes (Amersham Pharmacia Biotech), ECL Western blotting was performed under previously described conditions (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 39Choudhury P. Liu Y. Bick R.J. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) with a 1:1000 dilution of a polyclonal rabbit antiserum against a synthetic peptide homologous to the cytoplasmic tail of canine calnexin (StressGen) or a 1:5000 dilution of rabbit antiserum against the α-subunit of the 20 S proteasome (Calbiochem). Incubation with conjugated secondary antibodies, subsequent washings, and the detection of immunospecific bands were performed in a manner identical to that reported previously (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 39Choudhury P. Liu Y. Bick R.J. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Relative band intensities were quantified by standard densitometric analysis. Variants null(Hong Kong) and PI Z are retained in the ER of stably transfected hepatoma cells by distinct mechanisms (9Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1997; 272: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar), but each is subjected to intracellular disposal (23Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar). Intracellular turnover was examined in pulse-chase studies following a 15-min pulse with [35S]methionine. After 3 h of chase, 30% of pulse-radiolabeled null(Hong Kong) remained undegraded in the cell lysate (t12 = 120 min) (Fig.1, compare lanes 1 and2). Under identical conditions, the intracellular population of radiolabeled PI Z was not detected (t12 = 90 min) (Fig. 1, compare lanes 5 and 6), and only a fraction (∼15%) was secreted into the medium during this period (compare lanes 1 and 9), as previously reported (23Le A. Graham K.S. Sifers R.N. J. Biol. Chem. 1990; 265: 14001-14007Abstract Full Text PDF PubMed Google Scholar). No fraction of either radiolabeled variant was detected in the membrane pellet following cell lysis with Nonidet P-40; nor was the loss altered in response to the use of alternative polyclonal antisera for immunoprecipitation (data not shown). These findings indicate that the intracellular turnover of both variants was the result of intracellular degradation rather than a reflection of protein insolubility or hindered antibody recognition. Lactacystin, a specific irreversible covalent inhibitor of the multicatalytic proteasome (40Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1496) Google Scholar), arrested null(Hong Kong) turnover such that 95% of the pulse-radiolabeled molecules were detected in cells following a 3-h chase (Fig. 1, compare lanes 3 and4). In contrast, <5% of pulse-radiolabeled PI Z was detected in cells under identical conditions (Fig. 1, comparelanes 7 and 8), and this was not the result of accelerated secretion (compare lanes 9 and 10). In a separate set of experiments, not lactacystin, MG132, orN-acetyl-Leu-Leu-norleucinal, all inhibitors of proteasomal activity (28Rivett J.A. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (381) Google Scholar, 40Fenteany G. Standaert R.F. Lane W.S. Choi S. Corey E.J. Schreiber S.L. Science. 1995; 268: 726-731Crossref PubMed Scopus (1496) Google Scholar, 41Jensen T.J. Loo M.A. Pind S. Williams D.B. Goldberg D.L. Roirdan J.R. Cell. 1995; 83: 129-135Abstract Full Text PDF PubMed Scopus (770) Google Scholar), hindered PI Z turnover by more than 5% (data not shown). Overall, these findings indicate that variants null(Hong Kong) and PI Z are eliminated in hepatoma cells by distinct disposal pathways, the latter being somewhat more efficient. To initiate the biochemical description of the nonproteasomal disposal system, we explored the reported inhibitory effect of the metabolic poison sodium fluoride on PI Z disposal (24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar), since it fails to inhibit the intracellular turnover of variant null(Hong Kong) (39Choudhury P. Liu Y. Bick R.J. Sifers R.N. J. Biol. Chem. 1997; 272: 13446-13451Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). Since combinations of metabolic poisons are required to deplete intracellular ATP (43Braakman I. Helenius J. Helenius A. EMBO J. 1992; 11: 1717-1722Crossref PubMed Scopus (330) Google Scholar), we extended our investigation to test the hypothesis that the effect of sodium fluoride on PI Z disposal actually involves its reported role as an inhibitor of intracellular phosphatases (44Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2146) Google Scholar). Consistent with this notion, 80% of radiolabeled PI Z remained undegraded in cells following a 3-h chase in the presence of sodium pervanadate (Fig.2 a, lane 3), a membrane-permeable inhibitor of protein-tyrosine phosphatase activity (45Huyer G. Liu S. Kelly J. Moffat J. Payette P. Kennedy B. Tsaprailis G. Gresser M.J. Ramachandran C. J. Biol. Chem. 1997; 272: 843-851Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar), as compared with 20% under control conditions (lane 2). Pervanadate treatment had no demonstrable effect on null(Hong Kong) disposal under an identical set of conditions (data not shown). Importantly, PI Z turnover was inhibited to a similar extent as with sodium fluoride when pulse-radiolabeled cells were incubated with phenylarsine oxide (Fig. 2 b), an additional general tyrosine phosphatase inhibitor (46Singh S. Aggarwal B.B. J. Biol. Chem. 1995; 270: 10631-10639Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). In contrast, incubation with okadaic acid, a general inhibitor of serine/threonine phosphatase activities (47Cohen P. Holmes C.F. Tsukitani Y. Trends Biochem. Sci. 1990; 3: 98-102Abstract Full Text PDF Scopus (1259) Google Scholar), had no demonstrable inhibitory effect on PI Z disposal (Fig.2 b). Incubation with pervanadate arrested PI Z disposal without hindering the enhanced electrophoretic mobility of radiolabeled molecules in SDS-PAGE (Fig. 2 a), which reflects the removal of mannose from asparagine-linked oligosaccharides during intracellular retention (24Le A. Ferrell G.A. Dishon D.S. Le Q-Q. Sifers R.N. J. Biol. Chem. 1992; 267: 1072-1080Abstract Full Text PDF PubMed Google Scholar). This latter observation was common to all the tyrosine phosphatase inhibitors (data not shown), indicating that reversible tyrosine phosphorylation plays an important role at a relatively late step in the nonproteasomal disposal of variant PI Z. Selective permeabilization of the plasma membrane was performed after pulse radiolabeling with [35S]methionine (see “Materials and Methods”) as an alternate method to confirm that PI Z disposal occurs independently of the cytosolic proteasome. Quantitation after ECL Western blotting demonstrated that selective permeabilization of the plasma membrane had removed >95% of the immunoreactive proteasomal α-subunit (Fig.3 a). Under these conditions, only a negligible loss (∼20%) of pulse-radiolabeled variant null(Hong Kong) (Fig. 3 b, N) or wild type AAT (Fig. 3 b, M) was detected following 3 h of incubation in isotonic buffer. These findings indicated that the manipulation had arrested proteasome-mediated disposal without perturbing the structural integrity of the ER. In contrast, <5% of radiolabeled PI Z remained in the permeabilized cells under identical conditions (Fig. 3 c, Co). Moreover, the loss of radiolabeled protein was unaffected by lactacystin treatment (Fig. 3 c, Lct). At no period during the course of these experiments was radiolabeled PI Z detected in either the incubation buffer or membrane pellet of the Nonidet P-40 detergent lysate (data not shown). These data confirm that the loss of protein did not reflect its release from the ER or result from insolubility. Importantly, 82% of radiolabeled PI Z remained undegraded in permeabilized cells following a 3-h incubation when pervanadate was included in the incubation buffer (Fig. 3 c, Pv). These data indicate that the selective elimination of PI Z occurs independently of cytosolic factors. In a previous report (26Liu Y. Choudhury P. Cabral C.M. Sifers R.N. J. Biol. Chem. 1999; 274: 5861-5867Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), inhibitor studies were utilized to demonstrate how the processing of asparagine-linked oligosaccharides participates in the molecular capture of variant null(Hong Kong) for intracellular disposal. ER mannosidase I is the more abundant of two distinct mannosidases that can initiate the removal of mannose from asparagine-linked oligosaccharides (35Moremen K.W. Trimble R.B. Hersovics A. Glycobiology. 1994; 4: 113-125Cros" @default.
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