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• W1966403908 abstract "The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase β isoforms expressed in vitro and in Xenopusoocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins. The structural and molecular determinants that govern the correct membrane insertion and folding of membrane proteins are still ill-defined. By following the addition of sugar chains to engineered glycosylation sites (glycosylation mapping) in Na,K-ATPase β isoforms expressed in vitro and in Xenopusoocytes, in combination with biochemical techniques, we have defined the C-terminal end of the transmembrane domain of these type II proteins. N-terminal truncation and the removal of a single charged residue at the N-terminal start of the putative transmembrane domain influence the proper positioning of the transmembrane domain in the membrane as reflected by a repositioning of the transmembrane domain, the exposure of a putative cryptic signal peptidase cleavage site, and the production of protein species unable to insert into the membrane. Glycosylation mapping in vivo revealed that the degree of glycosylation at acceptor sites located close to the membrane increases with the time proteins spend in the endoplasmic reticulum. Furthermore, core sugars added to such acceptor sites cannot be processed to fully glycosylated species even when the protein is transported to the cell surface. Thus, the glycosylation mapping strategy applied in intact cells is a useful tool for the study of determinants for the correct membrane insertion of type II and probably other membrane proteins, as well as for the processing of sugar chains in glycoproteins. endoplasmic reticulum endoglycosidase H polyacrylamide gel electrophoresis binding protein leader peptidase l-1-tosylamido-2-phenylethyl chloromethyl ketone Subunit assembly of oligomeric membrane proteins often involves multiple but poorly understood interactions between the different subunits (for review see Ref. 1Geering K. Heijne G. Membrane Protein Assembly. Springer, New York1997: 173-188Google Scholar). Na,K-ATPase and H,K-ATPases are interesting model proteins for the study of functional roles of different subunit interaction sites, because these two enzymes are the only members of the cation-transporting P-type ATPase that are oligomeric and contain, in addition to the catalytic α subunit, a second subunit, the β subunit in the functionally active enzyme. Similar to most other P-type ATPases, the α subunits of Na,K- and H,K-ATPases are polytopic membrane proteins with 10 transmembrane segments that carry the main functional properties. The β subunits associated with Na,K- and H,K-ATPase α subunits are type II membrane proteins with a short cytoplasmic N terminus, a single transmembrane domain, and a large glycosylated ectodomain. To date, three Na,K-ATPase and one gastric H,K-ATPase β isoforms have been identified, which exhibit a similar domain structure but a low degree of sequence identity of 20–35%. At present, we know that β subunits have several functions that may be finely regulated by different isoforms. A primary role of the β subunit is to support the maturation of the Na,K- and H,K-ATPase α subunits, which, in contrast to other P-type ATPases, are stably expressed and become functionally active only when properly associated with a β subunit (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar). In addition to this chaperone function, the β subunit has also been shown to influence the transport properties of the mature Na,K-ATPase,e.g. its apparent affinities for K+ and Na+ (3Lutsenko S. Kaplan J.H. Biochemistry. 1993; 32: 6737-6743Crossref PubMed Scopus (124) Google Scholar, 4Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar, 5Eakle K.A. Kabalin M.A. Wang S.G. Farley R.A. J. Biol. Chem. 1994; 269: 6550-6557Abstract Full Text PDF PubMed Google Scholar, 6Shainskaya A. Karlish S.J.D. J. Biol. Chem. 1996; 271: 10309-10316Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 7Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Interaction sites that have been identified in Na,K- and H,K-ATPase α and β subunits involve the extracellular, transmembrane, and cytoplasmic domains. By using the two-hybrid system, an extracellular β domain adjacent to the transmembrane segment has been shown to interact with the extracellular loop between transmembrane segments M7 and M8 of the α subunit of Na,K-ATPase (8Colonna T.E. Huynh L. Fambrough D.M. J. Biol. Chem. 1997; 272: 12366-12372Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) and H,K-ATPase (9Melle-Milovanovic D. Milovanovic M. Nagpal S. Sachs G. Shin J.M. J. Biol. Chem. 1998; 273: 11075-11081Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). A β sheet-like structure formed by the 10 most C-terminal amino acids most likely represents another α-interaction site in the ectodomain of the β subunit (10Beggah A.T. Beguin P. Jaunin P. Peitsch M.C. Geering K. Biochemistry. 1993; 32: 14117-14124Crossref PubMed Scopus (39) Google Scholar). Interactions in the ectodomains of α and β subunits are important for the correct folding and the stabilization of the α subunit of oligomeric P-type ATPases (11Béguin P. Hasler U. Beggah A. Horisberger J.-D. Geering K. J. Biol. Chem. 1998; 273: 24921-24931Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 12Beggah A.T. Beguin P. Bamberg K. Sachs G. Geering K. J. Biol. Chem. 1999; 274: 8217-8223Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Furthermore, studies performed on chimeras formed between Na,K-ATPase β1 and gastric H,K-ATPase β subunits suggest that interactions with the β-ectodomain are responsible for the differences observed in the transport properties of the Na,K-ATPase associated with different β isoforms (5Eakle K.A. Kabalin M.A. Wang S.G. Farley R.A. J. Biol. Chem. 1994; 269: 6550-6557Abstract Full Text PDF PubMed Google Scholar, 7Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar). Evidence for interactions between transmembrane segments of the Na,K-ATPase α and β subunits has been obtained by cross-linking experiments (14Sarvazyan N.A. Modyanov N.N. Askari A. J. Biol. Chem. 1995; 270: 26528-26532Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 15Or E. Goldshleger R. Karlish S.J. J. Biol. Chem. 1999; 274: 2802-2809Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar), but the functional role of these interactions is not yet defined. Analysis of chimeric Na,K-ATPase/H,K-ATPase β subunits suggests that only transmembrane interactions of Na,K-ATPase β but not that of H,K-ATPase β subunits permit the correct folding and ER1 exit of the Na,K-ATPase α subunit (7Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 13Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar). Finally, the functional implications of subunit interactions in the cytoplasmic domains, which are supported by proteolysis protection assays (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar, 16Shainskaya A. Scneeberger A. Apell H.-J. Karlish S.J.D. J. Biol. Chem. 2000; 275: 2019-2028Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), are the least well understood. Indeed, truncation of the N terminus of Na,K-ATPase β1subunit does not impede α interaction and stabilization but significantly decreases the apparent K+ and/or Na+ affinity of the Na,K-ATPase (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar, 6Shainskaya A. Karlish S.J.D. J. Biol. Chem. 1996; 271: 10309-10316Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 7Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). However, results of a detailed mutational analysis indicated that the β N terminus may not be directly involved in the functional effects observed after complete N-terminal truncation (7Hasler U. Wang X. Crambert G. Beguin P. Jaisser F. Horisberger J.D. Geering K. J. Biol. Chem. 1998; 273: 30826-30835Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar), but rather that N-terminal truncation could indirectly affect another domain of the β subunit. To better understand the structural and functional roles of α-interaction domains in Na,K-ATPase β subunits, we aimed in this study to 1) define the membrane-spanning domain of the β subunit by identifying the amino acids that actually make up the transmembrane α-helix of Na,K-ATPase β subunits and 2) probe potential changes in the transmembrane domain after N-terminal truncation. To address these questions, we have applied a glycosylation mapping technique (17Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar). This assay is based on the observation that an engineered consensus glycosylation acceptor site can be modified by oligosaccharyltransferase only if this site is placed at a precise “minimal glycosylation distance” from a transmembrane segment (17Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar,18Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (114) Google Scholar). Therefore, the active site of oligosaccharyltransferase can be used as a reference point against which the position of membrane helices can be determined (18Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (114) Google Scholar). In this study, we apply for the first time the glycosylation mapping assay to proteins synthesized in intact cells, and our results show that the minimal glycosylation distance in intact cells is shorter than that of proteins synthesized in anin vitro translation system. Our studies also suggest that the C-terminal ends of the transmembrane helices of Na,K-ATPase β1 and β3 subunits are located near Leu58 and Met61, respectively. N-terminal truncation of β1 and β3 subunits results in a repositioning of the transmembrane helices relative to the membrane. Although these results do not resolve the question of the functional role of cytosolic α-β interactions, they clearly show that the N terminus of Na,K-ATPase β subunits is crucial for a correct β subunit topology that is compatible with proper assembly and, in consequence, the acquisition of the correct structural and functional properties of the Na,K-ATPase α subunit. The results also support our hypothesis that structural changes in the ectodomain and/or the transmembrane domain are responsible for the K+ effect observed in Na,K-ATPase associated with N-terminally truncated β subunits. Finally, our results validate the glycosylation assay applied in intact cells as a general tool to identify determinants of correct membrane insertion and, in addition, of the glycosylation processing of membrane proteins. Truncation and point mutants ofXenopus Na,K-ATPase β1 and β3isoforms contained in the pSD5 vector (19Good P.J. Welch R.C. Barkan A. Somasekhar M.B. Mertz J.E. J. Virol. 1988; 62: 944-953Crossref PubMed Google Scholar) were prepared by using the polymerase chain reaction method of Nelson and Long (20Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar). β1 subunits lacking 33 amino acids after the initiator methionine (β1t34, see Fig. 1) were prepared as described previously (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar). For the preparation of β3t37, lacking 36 amino acids after the initiator methionine, a β3 cDNA fragment was amplified using an antisense oligonucleotide consisting of nucleotides 301–320 tailed by primer D of Nelson and Long (20Nelson R.M. Long G.L. Anal. Biochem. 1989; 180: 147-151Crossref PubMed Scopus (294) Google Scholar) and a sense oligonucleotide comprising part of the noncoding sequence, the ATG coding for the first methionine, and the sequence coding for the amino acids Leu38 to Tyr43 of the β3 isoform. The amplified DNA fragment was then used as a primer to elongate the inverse DNA strand and was finally amplified using a sense oligonucleotide encoding part of the pSD5 vector and primer D of Nelson and Long. The mutated DNA fragment was introduced into the pSD5β1 vector using NheI and StuI restriction sites. N-Linked glycosylation acceptor sites were introduced in the β1 and β3 isoforms at various positions after the putative end of the transmembrane domain predicted by Kyte-Doolittle hydropathy analysis (21Kyte J. Doolittle R.F. J. Mol. Biol. 1982; 157: 105-132Crossref PubMed Scopus (16899) Google Scholar) (see Fig. 1). For this purpose, β1 fragments were amplified between an antisense oligonucleotide covering nucleotides 628–648 and tailed by primer D of Nelson and Long and sense oligonucleotides containing codons for the glycosylation acceptor site Asn-Ser-Thr. In these replacement mutants, Asn was placed at amino acid positions 59, 63, 65, 67, 70, and 74 for β1 glycosylation mutants −5, −1, +2, +6, +7, and +11, respectively (see Fig. 1). The mutated DNA fragments were introduced into the β1 cDNA lacking codons for the three natural glycosylation sites (22Beggah A.T. Jaunin P. Geering K. J. Biol. Chem. 1997; 272: 10318-10326Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) using NheI and BamHI restriction sites. The β1 glycosylation mutant +2 served as a template for the mutant Q56Lglyc+2 in which Gln56 of β1 was replaced by a leucine residue. N-Linked glycosylation acceptor sites were introduced in the β3isoform by first amplifying fragments of β3 cDNA using an antisense oligonucleotide consisting of nucleotides 301–320 and sense oligonucleotides containing codons for the glycosylation acceptor site Asn-Ser-Thr where Asn was placed at amino acid positions 65, 68, 70, 72, and 75 for β3 glycosylation mutants −1, +3, +5, +7, and +10, respectively (see Fig. 1). The mutated DNA fragments were introduced into the pSD5β3 vector usingNheI and StuI restriction sites. For the preparation of β1t34/K35L in which Lys35 of β1t34 was replaced by a leucine residue, a fragment of the β1 cDNA was amplified using β1t34 cDNA as a template, a sense oligonucleotide containing the point mutation, and the same antisense oligonucleotide used for the preparation of β1glycosylation mutants. For the preparation of β3t37/L38K in which Leu38 of β3t37 was replaced by a lysine residue, and of β3t37/L62A/T64G and β3t37/L62V/T64A, in which Leu62 and Thr64 were replaced by alanine and glycine residues, respectively, or by valine and alanine residues, respectively, a fragment of the β3 cDNA was amplified using β3t37 cDNA as a template, a sense oligonucleotide containing the point mutation, and the same antisense oligonucleotide used for the preparation of β3 glycosylation mutants. Chimera were constructed between the Xenopus Na,K-ATPase β1 subunit and Escherichia coli leader peptidase (Lep) (23Wolfe P.B. Wickner W. Goodman J.M. J. Biol. Chem. 1983; 258: 12073-12080Abstract Full Text PDF PubMed Google Scholar) by replacing the second Lep transmembrane domain (H2) by amino acid residues Gly27 to Thr61 of β1. This was performed by introducing BclI andNdeI restriction sites at nucleotides coding for amino acids 27 and 61 of β1 and amino acids 59 and 80 of Lep, which permitted the replacement of the BclI-NdeI Lep fragment with that of β1 subunits. The Lep cDNA contained N-linked glycosylation acceptor sites at positions +3, +16, and +20 from the putative end of H2 (17Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar). The nucleotide sequences of all constructs were confirmed by dideoxy sequencing. cRNAs coding for Bufo α1 (24Jaisser F. Canessa C.M. Horisberger J.D. Rossier B.C. J. Biol. Chem. 1992; 267: 16895-16903Abstract Full Text PDF PubMed Google Scholar),Xenopus β1 (25Verrey F. Kairouz P. Schaerer E. Fuentes P. Geering K. Rossier B.C. Kraehenbuhl J.-P. Am. J. Physiol. 1989; 256: F1034-F1043PubMed Google Scholar) and β3 (26Good P.J. Richter K. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9088-9092Crossref PubMed Scopus (92) Google Scholar) of Na,K-ATPases, Lep/β1 chimera, β-glycosylation mutants, and the molecular chaperone Bip (27Beggah A. Mathews P. Beguin P. Geering K. J. Biol. Chem. 1996; 271: 20895-20902Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar) were obtained by in vitro transcription (28Melton D.A. Krieg P.A. Rebagliati M.R. Maniatis T. Zinn K. Green M.R. Nucleic Acids Res. 1984; 12: 7035-7056Crossref PubMed Scopus (4042) Google Scholar). In vitro translation was performed using the Promega TnT Quick Coupled Transcription/Translation System kit. Briefly, 60 ng of cRNA was added to 20 μl of TnT Quick Master Mix, 2 μl of [35S]methionine (1000 Ci/mmol) at 10 mCi/ml (Amersham Pharmacia Biotech), and 0.3 μl of canine pancreatic microsomal membranes to give a final volume of 25 μl and was incubated at 30 °C for 90 min. In some cases, the proteins were subjected to endoglycosidase H (EndoH, Calbiochem) treatment as described (13Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar). Proteins were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and labeled proteins were detected by fluorography. Protein quantification was performed using a laser densitometer (KB Ultrascan 2202). Oocytes were obtained from Xenopusfemales as described (29Geering K. Theulaz I. Verrey F. Häuptle M.T. Rossier B.C. Am. J. Physiol. 1989; 257: C851-C858Crossref PubMed Google Scholar). Routinely, 7 ng of Na,K-ATPase α and/or 0.5–3 ng of β cRNA were injected into oocytes, and in some cases β and α cRNA were co-injected with 4 μCi/oocyte of Easy Tag Express (NEN Life Science Products). Oocytes not injected with [35S]methionine were incubated at 19 °C in modified Barth's medium containing 0.5 mCi/ml [35S]methionine. After a pulse of 4, 6, or 24 h, oocytes were subjected to a chase period of 24, 48, or 72 h in the presence of 10 mmcold methionine. Microsomes and in some instances digitonin extracts were prepared as described (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar), and the Na,K-ATPase α and β subunits were immunoprecipitated under denaturing or non-denaturing conditions as described (2Geering K. Beggah A. Good P. Girardet S. Roy S. Schaer D. Jaunin P. J. Cell Biol. 1996; 133: 1193-1204Crossref PubMed Scopus (125) Google Scholar) with a Xenopus β1(30Ackermann U. Geering K. J. Biol. Chem. 1992; 267: 12911-12915Abstract Full Text PDF PubMed Google Scholar), β3 (26Good P.J. Richter K. Dawid I.B. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9088-9092Crossref PubMed Scopus (92) Google Scholar), or Bufo α1subunit antibody (31Girardet M. Geering K. Frantes J.M. Geser D. Rossier B.C. Kraehenbuhl J.-P. Bron C. Biochem. J. 1981; 20: 6684-6691Crossref Scopus (435) Google Scholar). In some instances, the immunoprecipitates were subjected to EndoH treatment as described (13Jaunin P. Jaisser F. Beggah A.T. Takeyasu K. Mangeat P. Rossier B.C. Horisberger J.D. Geering K. J. Cell Biol. 1993; 123: 1751-1759Crossref PubMed Scopus (74) Google Scholar). The dissociated immune complexes were subjected to SDS-PAGE, and labeled proteins were detected by fluorography. Quantification of immunoprecipitated bands was performed with a laser densitometer (LKB Ultrascan 2202). To identify the cytosolic localization of the 35-kDa protein species produced in oocytes expressing β3t37 mutants, we followed its release into the medium after permeabilization of oocytes with saponin. For this purpose, oocytes were injected with wild type β3 or β3t37 mutant cRNA and labeled for 20 h with [35S]methionine. Twelve oocytes were incubated for 2 h at 19 °C in 500 μl of modified Barth's medium with or without 0.1% saponin before collection of the media and preparation of oocyte microsomes by centrifugation of the yolk-depleted homogenate at 20,000 × g for 30 min at 4 °C. Denaturing immunoprecipitations were performed with a β3 antibody on the total volume of the collected media and on microsomes corresponding to six oocytes. To test the protease sensitivity of the 28-kDa protein species produced in β3t37-expressing oocytes, we used two assays. In the first assay, oocytes were injected with wild type β3 or β3t37 mutant cRNA together with 4 μCi/oocyte of Easy Tag Express (NEN Life Science Products). After a 4-h pulse period, oocytes were homogenized with a plastic pestle in an Eppendorf tube in a solution containing 50 mm Tris-HCl (pH 7.5), 0.25 m sucrose, 50 mm potassium acetate, 5 mm MgCl2, and 1 mm dithiothreitol. Aliquots were incubated with 10 mm CaCl2 in the absence or presence of 0.2 mg/ml proteinase K (Merck) for 1 h at 4 °C before addition of 1 mg/ml phenylmethylsulfonyl fluoride. Immunoprecipitations were performed under denaturing conditions with a β3 antibody. In the second protease assay, oocytes were injected with wild type β3 or β3t37 mutant cRNA or with BiP cRNA and labeled for 24 h. Oocytes were then injected with 30 nl of H2O or 30 nl of a solution containing 25 mg/ml trypsin (TPCK-treated, Fluka). Oocytes were left for 1 h at 19 °C before preparation of digitonin extracts and immunoprecipitation with a β3 or a BiP antibody (27Beggah A. Mathews P. Beguin P. Geering K. J. Biol. Chem. 1996; 271: 20895-20902Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Na,K-pump activity was measured as the K+-induced outward current using the two-electrode voltage clamp method as described earlier (4Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar). Current measurements were performed 3 days after injection of oocytes with Bufoα cRNA together with different β cRNAs. To determine the maximal Na,K-pump current (I max), oocytes were loaded with Na+ in a nominally K+-free solution containing 200 nm ouabain, a concentration that inhibits endogenous Na,K-pumps but not the moderately ouabain-resistant exogenous Bufo Na,K-pumps (32Wang X.Y. Horisberger J.D. Mol. Pharmacol. 1996; 50: 687-691PubMed Google Scholar). The activation of the Na,K-pump current by K+ was determined in a Na+-free solution (140 mm sucrose, 0.82 mm MgCl2, 0.41 mmCaCl2, 10 mm N-methyl-d-glucamine-HEPES, 5 mmBaCl2, 10 mm tetraethylammonium chloride, pH 7.4), and the current induced by increasing concentrations of K+ (0.02, 0.1, 0.5, and 5.0 mm K+) was measured at −50 mV. To determine I maxvalues, the Hill equation was fitted to the data of the current (I) induced by various K+ concentrations ([K]) using a least square method, I =I max/(1 + (K12/[K])nH), whereK12 is the half-activation constant. According to previously published data (4Jaisser F. Jaunin P. Geering K. Rossier B.C. Horisberger J.D. J. Gen. Physiol. 1994; 103: 605-623Crossref PubMed Scopus (130) Google Scholar), the Hill coefficient was set to a value of 1.0. To define the transmembrane domains of Na,K-ATPase β1 and β3 isoforms and the possible changes in the transmembrane domain topology after N-terminal truncation, we have used a glycosylation mapping assay together with other biochemical techniques. For the glycosylation mapping assay, we have introduced Asn-Ser-Thr glycosylation acceptor sites at various positions around the predicted C-terminal ends of β1 and β3subunit transmembrane domains and have used the concept of minimal glycosylation distance defined as the number of amino acids separating the C-terminal end of the transmembrane domain from the first Asn residue that is half-maximally glycosylated (17Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar). Transmembrane domain predictions of β1subunits by various computer programs are shown in Fig.1 A. All programs predict that β1 contains a transmembrane domain α-helix, but prediction of its N- and C-terminal ends vary between programs. Kyte-Doolittle hydropathy analysis predicts that the β1transmembrane domain begins at Trp33 and ends at Ser63. With the exception of the HMMTOP program, predictions of the N-terminal end of the β1 transmembrane domain was similar with all programs and was located downstream of that predicted by Kyte-Doolittle analysis. Predictions of the C-terminal end of the β1 transmembrane domain ranged between Gly53 and Ser63. To determine experimentally the C-terminal end of the β1transmembrane domain, we first deleted the three natural glycosylation sites in the β1 lumenal domain and then introducedN-glycosylation acceptor sites (Asn-Ser-Thr) with Asn at positions −5, −1, +2, +4, +7, and +11 relative to Ser63,i.e. the C-terminal end of the β1transmembrane domain predicted by Kyte-Doolittle hydropathy analysis (see Fig. 1 A). Glycosylation of these proteins was first studied after in vitro translation in the presence of microsomes. About 80% of wild type Xenopus β1subunits containing the three natural glycosylation sites were glycosylated and migrated on SDS-polyacrylamide gels as a higher molecular mass species (Fig.2 A, lane 1) compared with β subunits lacking the natural glycosylation sites (lane 2). Glycosylation was absent in β1subunits containing a single engineered glycosylation site at position −5 (lane 3), but the proportion of glycosylated species gradually increased in β1 subunits containing single glycosylation sites at more distal positions and reached about 80% at position +11 (lanes 4–7, Fig. 2 D). As shown for the β1 glycosylation mutant +11 (Fig. 2 A,lane 8), all glycosylated species were sensitive to EndoH treatment, which specifically cleaves N-linked core sugars. Because half-maximal glycosylation occurs at a distance of about 10–11 amino acid residues away from the C-terminal end of natural, synthetic, or heterologous transmembrane domains introduced into the model membrane protein leader peptidase (Lep) (17Nilsson I.M. von Heijne G. J. Biol. Chem. 1993; 268: 5798-5801Abstract Full Text PDF PubMed Google Scholar, 18Nilsson I. Saaf A. Whitley P. Gafvelin G. Waller C. von Heijne G. J. Mol. Biol. 1998; 284: 1165-1175Crossref PubMed Scopus (114) Google Scholar, 33Nilsson I. Whitley P. von Heijne G. J. Cell Biol. 1994; 126: 1127-1132Crossref PubMed Scopus (124) Google Scholar), our results suggest that the β1 transmembrane domain is shorter than predicted by Kyte-Doolittle hydropathy analysis and ends around Leu58. To confirm this result, we also introduced the β1 transmembrane domain into the previously characterized Lep protein. For this purpose, we replaced the second transmembrane segment of Lep with the β1 transmembrane domain preceded by the last 6 cytoplasmic amino acids to ensure topological stability (Fig. 2 B). Wild type Lep containing a glycosylation site at position +20 from the end of the Lep transmembrane domain served as a control and was almost entirely glycosylated (Fig. 2 B,lanes 1 and 2). The Lep/β1 chimera containing a glycosylation site at position +3 (counting from the first Lep amino acid following the putative β1 transmembrane domain) was glycosylated by 30–40% (lanes 3 and4) and that containing a glycosylation site at position +16 was entirely glycosylated (lanes 5 and 6). The minimal glycosylation distan" @default.
• W1966403908 created "2016-06-24" @default.
• W1966403908 creator A5007682824 @default.
• W1966403908 creator A5020663562 @default.
• W1966403908 creator A5033100707 @default.
• W1966403908 creator A5074764709 @default.
• W1966403908 date "2000-09-01" @default.
• W1966403908 modified "2023-09-29" @default.
• W1966403908 title "Determinants of Topogenesis and Glycosylation of Type II Membrane Proteins" @default.
• W1966403908 cites W1516407458 @default.
• W1966403908 cites W1530502979 @default.
• W1966403908 cites W1541553752 @default.
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