Membrane Insertion of cy- and ,&Subunits of Na+,K+-ATPase

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1985 by The American Society of Biological Chemists, Inc

Vol. 260, No. 8, Issue of April 25, pp. 5154-5160,1985 Printed in U.S. A.

Membrane Insertion of cy- and ,&Subunits of Na+,K+-ATPase* (Received for publication, October 19,

1984)

Kathi Geering$, David I. Meyers, Marie-Pascale Paccolat, Jean-Pierre Kraehenbiihlll, and Bernard C.Rossier From the Institut de Pharmacologie, Universite de Lausanne,Rue du Bugnon 21, CH-1011 Lausanne, Switzerland, the IIrititut de Bwchimie, Universite de Lausanne, Chemin des Boveresses,CH-lo66 Epalinges, Switzerland, and the §European Molecular Biology Laboratory, 0-6900Heidelberg, Federal Republic of Germany

Insertion of the a- and #?-subunitsof amphibian epithelial Na+,K+-ATPaseinto pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic @-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes andto adopt a different pattern of N-linked core and terminal sugars in two differentamphibian species. The #?-subunitlacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of @-subunitassembled in vitro indicated a cleavable cytoplasmic domainofabout 2000 daltons. The catalytic 98-kilodalton a-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized @-subunit.In contrast to the @-subunit, membrane integration of the a-subunit was possible as late asa time point in itssynthesis which corresponded to about Vi-% of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the a-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both a- and #?-subunitoccurs during their synthesis but with a different time course.

and Edelman, 1976; Lo and Lo, 1980) and aldosterone (Gerring et al., 1982). This fact raises some interesting questions concerning the basic mechanism and subcellular location of synthesis andassembly of the two subunits into the functionally active enzyme. Agreement exists on the biosynthesis of the @-subunitwhich is translated on membrane-bound ribosomes cotranslationally inserted andcore-glycosylated in the endoplasmic reticulum (ER) and terminallyglycosylated during its transport to the cell surface (Fambrough, 1983; Fambrough and Bayne, 1983; Geering et al., 1982; Sabatini et al., 1982). On the other hand,considerable discrepancy exists in the results obtained on the biosynthetic pathway of the asubunit. Originally, Sabatini et al., (1982) reported that the a-subunit is synthesized by free polysomes and post-translationally inserted into membranes.The interesting hypothesis that a plasma membrane protein might follow such an unusual synthesis pathway is indeed supported by arecent publication (Hiatt et al., 1984) since a 96,000-dalton product was obtained by cell-free translation of mRNAfromfree polysomes. In addition, this polypeptide was not integrated into dog pancreas microsomes during its synthesis, as judged by its removal upon alkali treatment. It is important to point out that cotranslational insertion into the membranes is not the only mechanism by which polypeptides can be incorporated into membranes. TheoretiER, Golgi, or cally, post-translationaltranslocationinto plasma membrane is possible (Wickner, 1979) if appropriate The Na+,K+-ATPase’ (Anderson et at., 1982) is a mem- receptors existed in these membranes which would allow sitebrane-spanning oligomeric enzyme of the plasma membrane specific sequestration (Sabatini et al., 1982), as is the case, inmosteukaryotic cells (for review see Jmgensen, 1980; e.g. in mitochondria (for review see Hay et al., 1984). Along Kaplan, 1983; Katz, 1982). Its major function is the mainte- this line, Hiatt et al., (1984) proposed that the @-subunit nance of cellular Na+ and K+ homeostasis fundamental for might have such a receptor function and would anchor comnumerous cellular processes. T h e enzyme is composed of two pleted a-subunit in the ER membranes. Their hypothesiswas noncovalently linked subunits,a catalytic a-subunit (96,000based on the demonstration of a membrane-associated com116,000 daltons) and a glycosylated @-subunit(40,000-60,000 plex of 135,000 daltons recognized by anti-a antibodies. Howdaltons) of unknown function. Previous studies have shown ever, the proof for an a.@ complex could not be provided in that the biosynthesis of the two enzyme subunits is coordithis study for lack of a suitable anti-@ probe, and recently nately induced by hormones such as thyroid hormones (Lo several observations have been made by other authors which do not support the model of a post-translational @-subunit*This workwas supported by Grants 3636.80, 3.419.083, and 3.413.083 from the Swiss National Fund for Scientific Research. Part mediated membrane insertion of the a-subunit. Thus, Famof this workwas carried out a t the European Molecular Biology brough (1983)has shown that assembly of E- and @-subunits Laboratory at Heidelberg, Federal Republic of Germany. The costs into stoichiometriccomplexes occurs indeedvery rapidly but, of publication of this article were defrayed in part by the payment of as pointed out by the author, these results are not easily page charges. This article must therefore be hereby marked “aduerreconciled with the existence of two separate intracellular tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate pools of a - and @-subunits made on physically separated sites. this fact. In addition, preliminarydata indicate that both braina- and To whom correspondence should be sent. The abbreviations used are: ER, endoplasmic reticulum; Na+,K+- a+-subunitsare programmed by poly(A+) mRNA purified ATPase, (Na+ + K+)-dependent adenosine triphosphate phosphoryl- from bound polysomes and become membrane-associated durase (EC 3.6.1.3);ER, endoplasmic reticulum; SDS, sodium dodecyl sulfate; PMSF, phenylmethanesulfonyl fluoride; SDS, sodium dodecyl ing synthesis (Nabiet al., 1983). The mechanismof synthesis of the a-subunit thus remains sulfate; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulf0nicacid unsettled, and it isfor this reason that we have re-examined RM, rough microsomes.

+

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Membrane Insertionof Na+,K+-ATPaseSubunits

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search Inc., type 2). Highest A = 260 nm fractions werepooled, reprecipitated twice, and taken up in Hz0 at a concentration of 0.5 pg/,.tl. Size fractionation of poly(A+)was performed on linear 5-20% (w/v) sucrose gradients prepared in 10 mM Tris/HCl, pH 7.5, 1 mM EDTA, and 0.2% SDS. 150 pg of poly(A+) was loaded on 4.8 ml of sucrose gradients after heating for 3 min at 68 "C. Centrifugation was performed for 4 h at 45,000 rpm (SW 50 rotor) at 16 "C. The tube was then punctured from the bottom and 12 or 24 fractions were collected which were reprecipitated twice and mRNA content was determined a t A = 260 nm. mRNA was stored in water at a concentration of0.5 pg/pl at -80 "C. All glassware and solutions were autoclaved. Sucrose gradient fractions were tested in the reticulocyte lysate for their translational activity and immunoprecipitated with MATERIALS ANDMETHODS anti-a and anti-0 serum. Fractions enriched in mRNA coding for aCell Lines-A6 cells (derived from the kidney of Xenopus laeuis) subunit are designated as mRNAa and those enriched in mRNA and TBM cells (derived from the urinary bladder of Bufo marinus) coding for &subunit as mRNAP. mRNA coding for IgG light chain have been obtained from J. S. Handler, NationalInstitutes of Health, was purified from MOPC 41 mouse myeloma as described (Blobel Bethesda, MD. For review of culture conditions and characteristics and Dobberstein, 1975). of these epithelial cell lines, see Handler (1983). Cell-free Translations of Size-fractionated mRNA-Cell-free transBiosynthesis of Na+,K+-ATPuse in IntactCells-A6 or TBM cells lation of mRNAa or p were performed either in reticulocyte lysate (grown in 63 cmz Petri dishes) were labeled a t confluency with 150 (Amersham N90) or in wheat germ system prepared as described by pCi/ml [ ~ - ~ S ] m e t h i o n i (Amersham ne Corp.; specific activity >lo00 Warren and Dobberstein (1978) In preliminary experiments, optimal Ci/mmol) in 4 ml of tissue culture medium (HAM medium, Handler conditions for translation in the two systems were determined with et al., 1979) without serum and without methionine for 30 min to 4 h respect to RNA concentration and M e and K+ concentrations. at 28 "C. Incorporation of labeled precursor was stopped on ice by Routinely, 20 ng/pl mRNAa or mRNA/3from A6 or TBM cells or 10 addition of 6 ml of HAM containing 1 mM cold methionine and 20 ng/ml mRNA light chain were translated in a total volume of 25 or pg/ml cycloheximide. After scraping off the cells with a rubber policeman and two washes with HAM and 1 wash with Ringer's, cell 50 p1 in the presence of 140 mM K acetate and 1.2 mM Mg acetate extracts were prepared by boiling the cells directly in 4% SDS for 5 and 2.5-3 pCi/pl [35S]methioninefor 60 min at 30 "C in the reticulomin. Aliquots were precipitated with 10% trichloroacetic acid, and cyte lysate. In some experiments rough microsomes prepared from protein content (Lowry et al., 1951)and radioactivity were determined dog pancreas (Blobel and Dobberstein, 1975) were added during or after translation in the reticulocyte lysate at a concentration of 0.24 in the pellet after solubilization with 1 N NaOH. Immunoprecipitation of a- and @-subunitsfrom the cell extracts Amnm/50 pl lysate. At the end of translation, PMSF (2 mM)was (250 pgof protein) was performed as described below. In order to added to all samples which were then immunoprecipitated with antireveal the proteic structure of the &subunit, A6 or TBM cells were a or anti$ serum after solubilization with 3.5% SDS at 95 "C for 5 incubated for 18 h with 5 pg/ml tunicamycin (Calbiochem), an min as described below. Alternatively, samples were treated with 500 inhibitor of dolichol-dependent glycosylation (Elbein, 1981), before mM KC1 or alkali-treated (pH 11) by addition of '/s volume of 1 N cells were labeled for 45 min in the presence of the inhibitor, solubi- NaOH and left on ice for 15 min. The samples were then loaded onto lized, and immunoprecipitated with anti$ serum. Protease treatment a 600-p1 sucrose cushion (0.5 M sucrose, 20 mM Hepes, pH 7.4, 50 of cell homogenates was performed after cell labeling for 7 min with mM KCI) and centrifuged for 40 min at 2 "C at 49,000 rpm (SW 55, 400 pCi/ml [35S]methionine.Cells from 1 Petri dish (63 cm2) (about Sorvall rotor) before supernatants (neutralized with Ys volume of 1 N 2.8 mg of protein) were scraped and washed as described, taken up in HCI) and pellets were solubilized with SDS andimmunoprecipitated. 300 pl of Ringer's solution, and sonicated for 3 s with a Branson In some experiments, translation products from reticulocyte lysate sonifier (position 4). 1 0 0 - ~ 1aliquots were treated k0.2 mg/ml of were subjected to protease treatment before immunoprecipitation. trypsin (Sigma type XI) in the presence of 140 mM K acetate (to 200 pg/ml proteinase K (fungal, Merck) or 1-500 pg/ml trypsin were mimic ionic conditions of cell-free systems) for 60 min on ice. Protease added at the end of translation either in the absence or presence of digestion was stopped with a 5-fold excess (w/w) of soybean trypsin 1% Triton. After incubation for 60 min at 0 "C, proteinase K was inhibitor (Sigma) and left on ice for a further 10 min. The samples inhibited by a 2-fold excess (w/w) of PMSF and trypsin with a 5-fold were then centrifuged for 30 min at 100,000 X g at 4 "C, and the pellets were solubilized in 3.5% SDS at 95 "C for 5 min before excess (w/w) of soybean trypsin inhibitor. Samples were left on ice for an additional 10 min before centrifugation and/or immunoprecipimmunoprecipitation with anti-a serum. RNA Preparation from A6 and TBM Cells-RNA was prepared by itation. Synchronized Membrane Insertion-In order to determine the lata modification of the citric acid method (Schibler et al., 1980). 20 ml of membrane insertion of a- and p-subunitsof Na+,K+of 5% citric acid were added to 1 ml of frozen A6 cells or TBM cells est time points which were scraped on ice directly in 5% citric acid (final concentra- ATPase and ofIgG light chains,the following experiments were tion: 1 ml/Petri dish of 63 cm'). Homogenization was performed with performed. One large batch of reticulocyte lysate supplemented with 10 strokes at maximal speed with a glass-Teflon homogenizer on ice. [?3]methionine was preincubated for 2 min at 30 'C. After addition Homogenates were then centrifuged at 2,000 rpm in a SS 34 Sorvall of mRNAa or fi or mRNA light chain, translation proceeded up to 3 rotor for 5 min a t 2 "C. The pellets were discarded, and the superna- or 5 min. At this time point synthesis initiationwas stopped with 7tant was centrifuged at 15,000 rpm in the same rotor for 30 min at methylguanosine 5-phosphate (Na+salt, Sigma) (final concentration, 2 "C. The supernatant was carefully removed, the walls dried, and 4.5 mM) (Rothman and Lodish, 1977).At various time points, aliquots the pellet dissolved in 5 volumes/original cellvolume of dilution were takenand used for direct immunoprecipitation in order to buffer (2% SDS, 0.2 M Tris/HCl, pH 7.5, 5 mM EDTA, 200 pg/ml determine the time of completion of the various polypeptides (data heparin). If necessary, pH was adjusted to pH6-6.5. The same volume not shown). Other aliquots were added to roughmicrosomes and of chloropan, containing 1 volume of redistilled phenol, 1 volume of incubated up to 60-70 min at 30 "C. After this time, samples were chloroform + 0.05% (w/w) 8-OH-quinoline, and 1 volume of ANE either alkalinized and centrifuged or directly immunoprecipitated. buffer (10 mM Na acetate, pH 6, 100 mM NaCI, 1 mM EDTA), was Synthesis of a-subunit was completed around 60 min. Assuming a then added and vigorously shaken for 10 min at room temperature. After centrifugation, the aqueous phase was saved and the phenol molecular mass of 112/amino acid, the 98-kDa a-subunit contains and interphase re-extracted with dilution buffer. After centrifugation, about 875 amino acids and the synthesis rate can then be roughly the two aqueous phases were pooled and extracted three more times estimated to 15 amino acids/min. Immunoprecipitation and Detection of a- and P-Subunits-Immuwith chloropan. Total RNA was precipitated with 0.3 M Na acetate and 2 volumes of ethanol a t -20 "C overnight, centrifuged, and noprecipitation of a-subunit from A6 and TBMcells and of &subunit washed twice with 70% ethanol. The dried pellet was taken up in 10 from TBM cells was performed with antibodies prepared against mM Tris, 1 mM EDTA, pH 7.4, at a concentrationof 1pg/pl. Poly(A+) purified a- and &subunit from the kidney of B. marinus (Girardet et RNA was purified by passing total RNA (after heating for 2 min at al., 1981). Immunoprecipitation of @-subunitfrom A6 cells was per68 "C) twice over an oligo(dT)-cellulose column (Collaborative Re- formed with an antibody prepared against 8-subunit from the kidney

the question by studying the synthesis mode both in the intact cell and i n cell-free systems supplemented with dog pancreas microsomes. In addition, since we also had monospecific antibodies against the p-subunit at our disposition, we could follow the synthesis pattern or the a- and p-subunit in parallel. Our findings indicate that both /3- and a-subunits are inserted into ER membranes independently of each other during their synthesis. However, compared tothe P-subunit, a larger portion of a-subunit can be synthesized prior to association with the membrane.

Membrane Insertion of Na+,K+-ATPaseSubunits

5156

of X . leavis2since antibodies from B. marinus didnot cross-react with @-subunitfrom this species. Immunoprecipitation was performed as described (Geering et al., 1982) with the following modifications. After solubilization of the samples with 3.5% SDS a t 95 "Cfor 5 min, 11 volumes of 1% Triton in 50 mM Tris/HCl, pH 7.4, 5 mM EDTA, 150 mM NaCl, and 2 mM PMSF were added. The samples were then incubated with 5 pl of preimmune serum for 15 min on ice before addition of 50 pl of formalin-fixed Staphylococcus aureus (lo%, v/v). Incubation proceeded for an additional 20 min a t room temperature with shaking before centrifugation. The supernatants were transferred to new tubes, and cell-free translation products were immunoprecipitated with 10 pl and synthesis products from intact cells (250 pgof protein) with 100 pl of anti-or or anti-@serum. After overnight incubation at 2 "C and absorption of immunocomplexes to S. aureus, the immunoprecipitates were eluted with sample buffer at 95 "C for 5 min and loaded on 5-13% SDS-polyacrylamide gel electrophoresis gradients as described (Geering et al., 1982). In some experiments immunoprecipitates from biosynthetically labeled cell lysates were subjected to endoglucosaminidase H (Endo H, HealthService Inc.) treatment modified by Zilberstein et al. (1980). Two aliquots were precipitated with acetone for 1 h on ice, centrifuged, and the pellets resuspended in 50 mM Na acetate, pH 5.5, 2 mM PMSF either with or without 2milliunits of endoglucosaminidase H. After overnight incubation a t 37 "C, the samples were reprecipitated with acetone and taken up in sample buffer. Electrophoresis, fluorography of salicylate-treated (Chamberlain, 1979) and dried gels, and quantification by spectrophotometric scanning of autoradiographs were performed as described (Geering et al., 1982).

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RESULTS

Poly(A+) RNA Size Fractionation and Translational Actiuity-Poly(A+) RNA extracted from A6 and TBM cells and chromatographed on a column of oligo(dT)-cellulosewas sizefractionated on linear 5-20% sucrose gradients in order to enrich a- and @-subunit-encodingmRNA (Fig.1, upperpanel). Routinely, 12 or 24 fractions ofA6- or TBM-RNA were collected and tested for their translational activity in reticulocyte lysate. @-Subunitwas most efficiently synthesized from fractions containing 21-25 S mRNA species (Fig. 1, lower panel), and the increase in translational activity over total poly(A+) was about 15-fold in the highest enriched fraction of 23 S (data not shown). @-Subunitencoding mRNA, on the other hand, was contained in 13-17 S fractions (Fig. 1, lower panel). It cannot beexcluded that fractions enriched in mRNA@contain some residual mRNAa. On the other hand, however, it is clear form Fig. 1 that fractions of 23-25 S which contain mRNAa and which were used for the following studies on a-subunit synthesiswere free of mRNA@,(e.g. already in the 21 S fraction no more @-subunitcan be detected). This fact was of particular importance to study the significance of the concomitant synthesis of @-subunitfor the membrane insertion of the a-subunit. Corroboration of in Vitro and in Vivo Biogenesis and Membrane Insertion of a and @ Na+,K+-ATPase Subunits-Sizefractionated poly(A+) RNA isolated from A6 and TBM cells enriched for @-subunitmRNA, was translated in reticulocyte lysate in the presence and absence of dog pancreas microsomes. The immunoprecipitated translation products were compared with @-subunitsderived by immunoprecipitation of pulse-labeled cells. The nonglycosylated @-subunithas a molecular mass of 32 kDa (appearing as a doublet in A6 cells) and was observed as the product of immunoprecipitation in reticulocyte lysate translations (Fig. 2, lane 2 ) as well as in detergent-solubilized tunicamycin-treated A6 and TBM cells (Fig. 2, lunes 1 and 6 ) . This would indicate that @-subunit does not exist in a precursor form having a cleavable signal sequence. K. Geering, D. I. Meyer, M.-P. Paccolat, J.-P. Kraehenbuhl, and B. C. Rossier, unpublished data.

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98K

32K+

p-subunit

"subunit

FIG.1. Size fractionation of poly(A+) RNA from A6 cells (upper panel) and its translational activity for Na+,K+-ATPase subunits (lower panel). Upper panel, poly(A+) ( 0 - 0 ) or total RNA (-) purified from A6 cells were fractionated on a 510% sucrose gradient as described under "Materials and Methods." After centrifugation, 24 fractions were collected from the bottom of the tubes and the RNA content determined a t A = 260 nm. The sedimentation coefficient ( S ) of the mRNA contained in the various fractions was determined by using sized total RNA exhibiting two peaks of ribosomal RNA of 18 and 28 S and one peak of tRNA of 4 S. Lower panel, fractions of the sucrose gradient containing 13-25 S mRNA species were translated inreticulocyte lysate and immunoprecipitated with anti-or (fractions of 21-25 S) or anti-@(fractions of 1121 S) serum as described under "Materials and Methods." Shown are autoradiographs of the translation products obtained from different fractions of the sucrose gradient. 32K, 32,000, for example. In the case of @-subunitlabeled by a 30-min pulse in uiuo, or in acell-free system supplemented with rough microsomes, the core-glycosylated formof 40 and 42 kDa was observed in A6 and TBM cells, respectively (Fig.2, lunes 3, 4, 7, and 10). These peptides were sensitive to endo-@-N-acetylglucosaminidase H treatment (data not shown), a fact which indicates that core-glycosylation of the @-subunitis N-linked and of the high-mannose type (for review see Hubbard et al., 1981). Four-hour pulse labeling yielded the terminally glycosylated

Membrane Insertion of Na+,K+-ATPase Subunits

1

2

3

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A6 CELLS

TBM CELLS

FIG.2. Biosynthesis and processing of Na+,K+-ATPase @-subunit in intact cells and in a cell-free system. Lanes I, 3, 5, and 6-9, synthesis products ofA6 or TBM cells labeled with [%]methionine. The cells were lysed with 3.5% SDS, and immunoprecipitation was performed with anti-@serum. Lanes 1 and 6, cells were preincubated for 18 h with 5 pg/ml tunicamycin and thenpulse-labeled for 45 min in the presence of the inhibitor; lanes 3, 5, 7-9, no treatment with tunicamycin; lanes 3 and 7, 30 min pulse; lanes 5, 8, and 9, 4-h pulse; lane 9, immunocompetition with purified @-subunit.Lanes 2, 4, and 10, synthesis products of mRNAP from A6 or TBM cells translated in reticulocyte lysate in the absence ( l a n e 2) or presence (lanes 4 and 10) of rough microsomes and immunoprecipitated with anti-@serum; lanes I 1 and 12, an aliquot of the sample shown in lane 10 was subjected to proteolysis with 200 pg/ml trypsin for 60 min a t 0 "Ceither in the absence ( l a n e 11) or presence (lane 12) of 1% Triton. Trypsin digestion was stopped by addition of a 5-fold excess (w/w) of soybean trypsin inhibitor before immunoprecipitation with anti-@serum. Details appear under "Materials and Methods." 49K, 49,000, for example.

subunit which had a molecular mass of 49 kDa (or -140 kDa in a trimeric form) in A6 (Fig. 2, lane 5 ) and of 60 kDa in TBM cells (Fig. 2, lune 8 ) and which were endoglucosaminidase H-resistant (data not shown). As shown in Fig. 2, lane 9, the immunoprecipitation of all the glycosylated species of TBM cell @-subunitcould be immunocompeted by the addition of purified @-subunit derived from the kidney of B. marinus. As a criterion for membrane insertion, @-subunit synthesized in thepresence of RM was digested with trypsin.As can be seen in Fig. 1 (compare lanes 10, 11, and 12), all but 2000 daltons of the @-subunitwas protected from proteolytic digestion. The same observation was made withproteinase K digestion (data notshown). These dataindicate that themain portion of @-subunit is integrated in or translocated across the ER membraneduring its synthesis and that asmall domain is exposed to the cytoplasmic face in the core-glycosylated form of the @-subunit. A6 cells were pulsed with [35S]methioninefor 7 min, sonicated, and the membrane and soluble fractions isolated by centrifugation. The 98-kDa a-subunit which was immunocompeted by purified a-subunit (Fig. 3, lanes A and B ) was recovered (>95%) in the membranous pellet (compare Fig. 3, lanes A and C). This material could be digested by exogenously added protease to a new membrane-associated form of 34 kDa (Fig. 3, lane D),which could be immunocompeted by the addition of purified 98-kDa a-subunit (Fig. 3, lane E ) . Experiments conducted in the reticulocyte lysate system also produced a 98-kDa a-subunit (Fig. 3, lane I), immunocompetable with purified a-subunit (Fig. 3, lane 2). The 98kDa peptide, translated in vitro in theabsence of membranes, appears to be in a soluble form, as little of this species could be pelleted at 200,000 x g for 40 min (Fig. 3, lane 3, S P). Translations performed in thepresence of RM also produced

+

a protein of 98 kDa. It appears that the a-subunit does not undergo cotranslational modifications such as a cleavage of a signal sequence. However, it is also possible that our gel system does not permit us todetect smallchanges in molecular weight for proteins of this size. After treatment of RM used in the cell-free system with alkali (pH 11) or high salt to solubilize all but integral membrane proteins, greater than 95% of the newly synthesized a-subunit could be recoveredin the membrane pellet (Fig.3, lane 4, S + P). Since these translations have been performed with highly enriched mRNAa fractions devoid of mRNA@,these results indicate that stable membrane integration of a-subunit has occurred independently of the presence of newly synthesized P-subunit. As a control for the integrity of rough microsomes during translation and for the efficiency of the subsequent alkali treatment, a well-characterized secretory protein (IgG light chain) (Meyer et al., 1982) was translated in parallel in the presence of RM, and thesamples were subjected to the same procedure as for a-subunit aftertranslation. In thiscase, the mature form ofIgG lightchain with its signal sequence cleaved was exclusively recovered from the supernatant, indicating that theRM used were indeed competent for preprotein processing and that alkali treatment opened up the membrane vesicles thereby releasing secreted products as expected (data notshown). Upon proteolysis, membranes which had been incubated in the presence of mRNAa (obtained from A6 cells) yielded a polypeptide of 34-kDa (Fig. 3, lanes 6 and 7) as in thecase of a-subunit synthesized in vivo (Fig. 3, lane D).In addition, when membranes were added late during cell-free translation (45 min)more than 95% of the 98-kDa material was recovered in the supernatant of a pH 11-treated preparation (Fig. 3, lane 5, S P).These data indicate that themode of membrane insertion of a-subunit is comparable in vivo and in vitro and

+


5158

98 K 4-

J+

34K +

I

P

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S

I

P

1

1

1

S

1

P

1

S

1

P

1

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FIG. 3. Biosynthesis and membrane insertion of Na+,K+-ATPasea-subunit in intact cells and in a cell-free system. Lanes A-E, A6 cells were labeled for 7 min with [35S]methionine,scraped, washed, and sonicated in an isotonic medium for 3 s as described under “Materialsand Methods.” One of two equal aliquots was subjected to proteolysis with 200 pg/ml trypsin (9 mg of protein/ml) in presence of 140 mM K acetate and the treated and nontreated samples were left on ice for 60 min. Proteolysis was stopped by addition of a 5-fold excess (w/w) of soybean trypsin inhibitor and further incubated for 10 min on ice. The samples were then centrifuged at 100,000 X g for 30 min and the pellets (P) and supernatants (S) solubilized in 3.5% SDS by heating at 95 “C for 5 min before immunoprecipitation with anti-a serum as described under “Materials and Methods.” Lanes A-C, immunoprecipitates obtained from nondigested cell homogenates; lanes D and E , immunoprecipitates obtained from trypsin-treated cell homogenates; lanes B and E , immunocompetition with purified a-subunit; lanes 1-7, immunoprecipitates obtained with anti a-serum from reticulocyte lysates supplemented with mRNAa from A6 cells; lanes 1-3, translation in the absence of RM; lane 2, immunocompetition with purified a-subunit; lanes 3 S + P, after translation, the lysate was adjusted to pH 11 by addition of 1 N NaOH and centrifuged on a 0.5 M sucrose cushion. Immunoprecipitation was performed on the supernatant(S)after neutralization with 1N HCl and on the Lanes 4 S + P, translation in presence ofRM. After translation, lysates were alkali-treated and pellet (P). centrifuged on a 0.5 M sucrose cushion and P and S immunoprecipitated. Lunes 5 S + P, RM were added after 45 min of translation and incubation continued up to 60 min. Alkalinization and centrifugation were performed as above before immunoprecipitation of S and P. Lanes 6 and 7, after translation in the presence of RM, one of two aliquots was subjected to proteolysis with 50 Fg/ml of trypsin for 60 min a t 0 “C. After addition of a 5-fold excess (w/w) of soybean trypsin inhibitor, the samples were centrifuged on a 0.5 mM sucrose cushion before immunoprecipitation of P. Lane 6,without trypsin; lane 7, plus trypsin. Details appear under “Materials and Methods.”

that membranes must be present prior to 45 min of translation for proper integration to occur. Time-dependence of Membrane Addition for Insertion of in Vitro Synthesized a- and /?-Subunits-In order to determine up to which point in the translation membrane insertion can still occur, RM was added at various time points to a synchronized in vitro translation system. In the case of a-subunit, the 98-kDa peptide was entirely recovered in a pH 11-resistant membranous form when RM was added prior to 15-25 min of translation (Fig. 4A).At this time point %-‘/z of the polypeptide was synthesized (for details see “Materials and Methods.”). Addition of RM at later time points led to a progressive decrease in the amount of membrane-associated material concomitant with an increase in the soluble form recovered from the supernatant of pH 11treated and centrifuged samples (Fig. 44).Addition of RM at later time points led to increasingly less pH11-resistant material. The point at which only half of the material translated became alkali-resistant corresponded to 33-40 min (tlIz).This point would indicate that about 490-600 amino acids of a-subunit were already polymerized. In the case of /?-subunit or IgG light chain, membrane insertion or translocation began to decrease rapidly from the earliest analysis points onward (Fig. 3, B and C ) . Loss of membrane insertion was followed by the increasing appearance of the nonglycosylated or the precursor form of the /?subunit andthe IgG light chain, respectively. tllzcorresponded to 13 min in the case of the @-subunit and to 7 min in the case of IgG light chain, which means that about 195 and 105 amino acids were polymerizedof the respective polypeptides. These data demonstrate that theinsertion of a-subunit can

occur later in translation than in the other characterized systems such as /?-subunit or IgG light chain. What is clear from these data andFig. 2, however,is that in the experimental system used, a-subunit cannot be inserted into RM in a post-translational manner. Between 50 and 60 min of translation, the time needed for in vitro synthesis of the complete 98-kDa form, virtually 100% of the peptide synthesized was not membrane-associated (pH 11-soluble). DISCUSSION

Several lines of evidence indicate in thisstudy that both @and a-subunits of Na+,K+-ATPase are cotranslationally integrated into ERmembranes but probably by different kinetics. In particular, the @-subunit translated on bound polysomes (Sabatini et al., 1981/1982) behaves like many characterized membrane and secretory glycoproteins. Our data indicate that the early stepsinits biosynthesis are indeed characterized by a rapid interaction with and translocation across the ER membranes with concomitant N-linked coreglycosylation. On the other hand, the membrane association of the a-subunit, although cotranslational as in the case of the @-subunit,can still occur after a considerable polymerization of amino acids which amounts to about 40% of completion of the 98-kDapolypeptide. In this respect, the asubunit resembles the erythrocyte band I11 (Braell and Lodish, 1982) and theglycoprotein of corona virus (Rottier et al., 1984) which also becomes inserted into membranes late in their translation.Hiatt et al. (1984) haverecently investigated the membrane assembly of Na+,K+-ATPase a-subunit. In agreement with our data, no cotranslational modifications of the a-subunitsuch as signal sequence cleavage could be dem-


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the &component in the 135-kDa form for lack of a suitable antibody. We have also observed a similar pH 11-resistant high molecular component in some of our preparations, but its appearance was limited to anti-aimmunoprecipitations of proteins translated in the wheat germ system primed with pfree, mRNAa. Importantly, it was not immunocompeted by excess 98-kDa a-subunit.' In addition, our data obtained in the reticulocyte lysate clearly indicate that membrane insertion of a-subunit in its 98-kDa form can occur in the absence of mRNA coding for P-subunit. The fact that the results of Hiatt et al. (1984) indicate a large soluble pool of nonintegrated 96-kDa a-subunit is difficult to reconcile with our finding of nearly quantitative membrane insertion of material translated invitro. A possible explanation could be that the microsomes in the two studies differed in their efficiency of nascent peptide insertion or translocation. In our system, controls were included to indicate that the RM used were fully capable of inserting and processing both &subunit aswell as IgG light chain. The finding that a-subunitis cotranslationally inserted into rough microsomes,although possibly later than proteins with N-terminal insertion signals, implies that mRNA coding for a-subunit should be membrane bound. This contradicts the original observations that a-mRNA is derived from a pool of free polysomes (Hiatt et al., 1984; Sabatini et al., 1981/1982). The fact that insertion may occur relatively late in translation could lead to a weakened interaction between polysomes and ER by virtue of fewer ribosomes having already translated the appropriate peptide length needed for insertion at any TIME (x MEMBRANEAWlTlON (minutes) given time. Such a notion could also explain the finding of FIG. 4. Synchronized membrane insertion of Na+,K+ATPase subunits and of IgG light chain. A, time course of about 5-10% of cytoplasmic a-subunit in pulse-labeled intact membrane insertion of a-subunit. mRNAa from A6 cells was trans- cells, including A6 cells in the present study, MDCK cells lated in reticulocyte lysate for 5 min before addition of 7-methylgua- (Hiatt et al., 1984), as well as toad bladder cells (Rosier, nosine 5-monophosphate to a final concentrationof 4.5 mM. Aliquots 1984) if one assumes that a fraction of this protein may be were added a t different times to RM and further incubated up to 70 completed without having the opportunity to interact with min. Samples were then alkali-treated to pH 11 and centrifuged on a 0.5 M sucrose cushion. Shown are percentages of immunoprecipitable intracellular membranes. An alternative explanation has rematerial with anti-a serum at different times of membrane addition cently been given by Caplan et al. (1983) who suggestedthat from either supernatants (0)or pellets (0)quantitated by densitom- the soluble cytoplasmic pool might be a mere artifact of harsh etry. B , time course of membrane insertion of 0-subunit. mRNAP cell lysis procedure. It can, however, not be excludedthat two from A6 cells was translated in reticulocyte lysate for 3 min before different pools of a-subunit exist in the intact cell which are addition of 7-methylguanosine 5-monophosphate (final concentration 4.5 mM). Aliquots were added at different times to RM and further made on different sites and/or which are distributedin differincubated up to 60 min. Samples were then immunoprecipitated with ent cellular compartments. It is also worth mentioning in this anti-0 serum. Shown are percentages of nonglycosylated 0-subunit at context that cotranslational insertion of a-subunit cannotbe different timesof membrane addition as quantitated by densitometry. unambiguously proven in the absence of verifiable cotranslaC, time course of membrane translocation and processing of IgG light tional modifications such as core-glycosylation or signal sechain. mRNA light chain was translated in reticulocyte lysate for 3 min before addition of 7-methylguanosine 5-monophosphate (final quence cleavage. Thus, although insertion of a-subunit occurs concentration 4.5 mM). Aliquots were added a t different times to RM late during its translation in a cell-free system, this may not and further incubated up to 60 min before SDS-gel electrophoresis. correspond to the in vivo situation. Our finding that a very Shown are percentages of prelight chain at different times of mem- early synthetic product of the a-subunit yielded the same brane addition quantitated by densitometry. proteolytic fragment in vivo and in vitro supports, however, the notion that the cell-free system accurately reflects the onstrated. It is clear, however, that this result needs confir- situation in the intact cell. mation by N-terminal sequencing. Moreover, we confirm their One way tofurther define the mannerand location of finding that a- and @-subunits are encoded by distinct mRNA membrane association willbe to study the behavior of apopulations. In several respects, however, our results disagree subunit in the wheat germ system. It is in this translation with those of Hiatt et al. (1984). We have indeed found that system where dependence on the signal recognition particle the 98-kDa subunit is cotranslationallyinsertedinto ER can be determined. Signal recognition particle has been shown membranes in contrast to the 96-kDa polypeptide of their to be essential for the cotranslational insertionof a variety of translations which was never found associated with mem- secretory and membrane proteins (Anderson et al., 1982,1983; branes. A larger 135-kDa polypeptide was observed in their Meyer et ~ l . 1982; , Meyerand Dobberstein, 1980; Walter and study as the only alkali-resistant form. They postulated that Blobel, 1981) and may be a useful tool to discriminate methis species might represent an a . @ complex since it was only chanistic differences between a- and @-subunitinsertion. produced when the twomRNA populations coding for asubunit and smaller peptides, respectively, were translated Acknowledgment-We would like to thankS. Perret-Gentil for her simultaneously. Unfortunately, the authorscould not identify skillful technical assistance.

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