THE JOURNAL OF BIOLOGICAL CXEM~STRY Vol. 265, No. 19, Issue of July 5, pp. 10829-10835,199O 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Cell-free Transcription and Translation of Na,K-ATPase a! and @ Subunit cDNAs*

(Received for publication, December 11, 1990)

Eftihia Cayanis, Hagen BayleyS, and I. S. Edelman From the Department of Biochemistry & Molecular Biophysics of tti College of Physicians & Surgeons of Columbia University, New York, New York 10032

Synthetic mRNAs (i.e. cRNA, and cRNAJ were ob- tained by cell-free transcription of Ml3 KS(+) (Blue- script) expression vectors which contained the entire coding region of the (Y or @ subunits of lamb kidney Na,K-ATPase. Translation in reticulocyte lysates of cRNA, yielded full length (Y polypeptide, as well as a limited array of immunoprecipitable lower molecular weight products. cRNAB yielded a single immunopre- cipitable full length polypeptide.

Association of the (r polypeptide with the microsomal membranes was obtained only co-translationally. Fif- teen to 60% of the membrane-associated (Y subunit was resistant to extraction with alkali. The resistance of a 29-kDa fragment to trypsinolysis indicated that the (Y subunit was inserted into microsomal membranes. In the presence of dog pancreatic microsomes, the j3 poly- peptide was glycosylated as indicated by the appear- ance of three higher molecular weight polypeptides that were sensitive to endoglycosidase H and bound to Concanavalin A. The @ subunit was predominantly translocated into the lumen of the endoplasmic reticu- lum since 90% of the mass of the membrane-associated fi polypeptide was resistant to trypsin (i.e. reduced in size from 40 kDa to 37.6 kDa), and 95% of all of the #I chains were resistant to extraction with alkali. Neither the a nor the B subunits have NH&erminal leader signal sequences, but both may require the signal rec- ognition receptor for membrane insertion, as evi- denced by inhibition of incorporation of both subunits into microsomes pretreated with iV-ethylmaleimide. Simultaneous translation of cRNA, and cRNAB did not enhance membrane insertion of either the (Y or /3 poly- peptide.

Na,K-ATPase’ is an integral membrane protein consisting of a large catalytic (Y subunit that is noncovalently linked to a smaller heavily glycosylated @ subunit (Kyte, 1972; Peterson

*Financial support for this project was provided by National Institutes of Health Grants CA22376 and AM36618. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

$ Present address: Worcester Foundation for Experimental Biol- ogy, 222 Maple Ave., Shrewsbury, MA 01545.

i The abbreviations used are: Na,K-ATPase, (Na+ + Kf)-depend- ent adenosine triphosphate phosphohydrolase (EC 3.6.1.37); DTT, dithiothreitol; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PBSE, phosphate-buffered saline containing EDTA, PMSF, phenvlmethanesulfonvl fluoride: NP40. Nonidet P-40: SDS-PAGE. sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NEM, N: ethylmaleimide; SRP, signal recognition protein; bp, base pair(s); kb, kilobase pair(s).

and Hokin, 1981; Jorgensen, 1974 and 1982). The primary structures of both (Y and /3 subunits, including the three (Y isoforms (designated (Ye, CQ, and (Ye), have been deduced from nucleotide sequences of cDNAs from a variety of species and tissues (Shull et al., 1985,1986a, 1986b; Schneider et al., 1985; Mercer et al., 1986; Shull and Lingrel, 1986; Kawakami et al., 1985 and 1986; Noguchi et al., 1986; Ovchinnikov et al., 1986 and 1987). Based on these sequences, the /3 subunit contains -305 amino acids depending on species (i14~ - 35,000 without the carbohydrate), only a single transmembrane domain of approximately 28 uncharged amino acids near the NH2 ter- minus, a small cytoplasmic domain that has been mapped to the hydrophilic NHz-terminal segment of l-33 amino acid residues, a large extracellular surface COOH-terminal domain comprising the last 242 amino acid residues, and no hydro- phobic NH*-terminal leader signal sequence (Shull et al., 1985; Mercer et al., 1986; Kawakami et uZ., 1986; Noguchi et al., 1986; Takeyasu et al., 1987).

The CQ subunit contains -1020 amino acids and spans the membrane several times (Shull et al., 1985 and 1986b; Ka- wakami et al., 1985; Ovchinnikov et al., 1986; Herrera et al., 1987). The hydrophilic NH, and COOH termini are cyto- plasmic, as are the phosphorylation (aspartyl-PO1) and fluo- rescein isothiocyanate binding sites that form part of the ATP binding site (Bastide et al., 1973; Farley et al., 1984; Kirley et al., 1984; Shull et al., 1985; Xu, 1989).

Coordinate synthesis and degradation of the a: and p sub- units of the mature enzyme have been inferred from amino acid labeling studies in rat kidney and eel electroplax (Lo and Edelman, 1976; Churchill and Hokin, 1979; Lo and Lo, 1980). The mechanism mediating coordinate biogenesis of the sub- units, however, has not yet been elucidated. Based on cell- free translation of poly(A+) RNA fractions, Hiatt et al. (1984) proposed that the guinea pig renal (Y subunit may be post- translationally integrated into microsomal membranes by binding to a co-translationally integrated p subunit or a related peptide. Similar studies on translation of poly(A+) RNA fractions, from rat kidney, toad kidney, and chicken muscle cells in culture, however, yielded evidence of inde- pendent, co-translational membrane integration of the (Y and fi subunits (Geering et al., 1985; Akayama et al., 1986; Tamkun and Fambrough, 1986).

Since the (Y- and /3-mRNAs are present in very low abun- dance (probably less than 0.2%) in poly(A+) RNA fractions, the earlier studies may have reflected variations in the com- position of the various RNA fractions derived from a multi- plicity of tissues and species. To resolve these discrepancies, a further analysis of the translational mechanisms involved in the biogenesis of the LY and p subunits was undertaken with full length lamb kidney cRNAs ((u and /3), derived by cell-free transcription, translated in reticulocyte lysates.

10829

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10830 Transcription and Translation of Na,K-ATPase

a. x:7 bp I 1

Eogl Sac11 175bP 3347 bp

t 3

t 3’

PO63 bp/ < 254bp 35Obp

B 2579bp

I I Smol Cl01

460 bp 1957 bp

5’ 1

3’ 52Bbp 909bp 1142b,,

3172 bp 3’S5’ Sdl EN

sac II Eogl T3

3’ I+cutctt( 5’

T, polymrose mRNAo

a ‘497bp 3,

SnWl Cl01

T3 T7 Smol Ch ---I 3’

FIG. 1. Construction of cDNA expression vectors for in vi- tro transcription of Na,K-ATPase a (left) and p (right) sub- units. a, full length coding regions of cDNA,, and cDNA,,. The 5’. and X,-untranslated regions were truncated. Coding sequences are indicated by the thick bars. b, restriction map of cDNA (n and 6) in pBR32.2 vector. c, insertion of cDNA (tr and 8) into Ml3 KS(+) (Hluescript) vector. d, linearization of recombinant MI3 KS(+) to produce template for transcription of the cDNAs. Restriction endo- nuclease sites used in the construction of these vectors as well as the size of inserts are as shown.

MATERIALS AND METHODS2

RESULTS

Construction of the Expression Vectors-Full length cDNA,, (3667 bp) was excised from the recombinant pBR322 by digestion with EugI and Sac11 (Fig. 1). The recovered 3.17-kb fragment containing the entire coding region of cDNA,,, 79 bp of untranslated 5’-sequences, and 30 bp of untranslated 3’-sequences was ligated, in the correct orientation into Ml3 KS(+) (the transcription vector) (Fig. 1). The removal of 175 bp from the 5’-end and 320 bp from the 3’-end of the original insert eliminated the possibility of internal hybridization of the poly(G)/poly(C) tails in cRNA,, transcripts. Restriction mapping of minipreps identified the correct 6.1-kb recombi- nants (i.e. 3.17 kb of cDNA,, plus 2.95 kb of Ml3 KS(+)).

The recombinant pBR322 that contained the 2.6-kb cDNA,, insert was cut with SmaI, ClaI, and PvuI, and 1.5 kb of the insert was ligated into Ml3 KS(+) (Fig. 1). The correct recombinant (4.5 kb; 1.5 kb of cDNA,~ plus 2.95 kb of Ml3 KS(+)) was identified by restriction analysis. The inserted 1.5-kb fragment contained 68 bp of the 5’-untranslated region, 520 bp of the 3’-untranslated region, and the entire 909 bp of the coding region (Fig. 1).

In Vitro Transcription-The recombinant Ml3 KS(+)-con- taining cDNA,. was linearized with SacII, and the cDNA,, plasmid with C&I. T,c RNA polymerase was used to transcribe cDNA,. and T; RNA polymerase to transcribe cDNA,< (Fig.

‘The “Materials and Methods” are presented in miniprint at the end of this paper. Miniprint is easilv read with the aid of a standard magnifying glass. Full size photocopies are included in the microfilm edition of the .Journal that is available from Waverly Press.

1). Single cRNA,, and cRNA,, products of 3.1 kb and 1.5 kb, respectively, were obtained with these templates, as shown by 1% agarose gel electrophoresis (data not shown). In prelimi- nary experiments, the mRNAs were capped with m’G(5’) ppp(5’)G. The capped and uncapped mRNAs were translated with equal efficiencies, however (data not shown). Accord- ingly, the cRNA products were not capped for the remainder of the experiments.

In Vitro Translation-cRNA,, and cRNA,,, obtained by in vitro transcription, were translated in a nuclease-treated rab- bit reticulocyte system (Fig. 2). cRNA,~ yielded a major band with a M, of 34,000 and a minor band of 30,000 (Fig. 2, lane 6). The translational products of cRNA,,, however, were het- erogeneous. The major bands had apparent M, of 92,000, 82,000, 76,000, and 69,000 (Fig. 2, lane 4).

To assess whether the lower M, peptides (82,000, 76,000, 69,000) were incomplete chains still bound to ribosomes, the released chains were characterized. Centrifugation of the translation mixtures at 149,000 x g for 30 min to separate the ribosome-bound from the free polypeptides did not alter the electrophoretic patterns of the N products (data not shown).

Full length lamb N, peptide has a M, of 112,177 computed from cDNA,,, (Shull et al., 1985). In SDS-PAGE, however, mature N subunits migrate at 90 to 100 kDa (Lane et al., 1979; Peterson and Hokin, 1981; Collins et al., 1983). To assess whether the o( product that migrated at 92 kDa (Fig. 2) was essentially full length, mature purified lamb and dog kidney (Y] was analyzed by 7.5% SDS-PAGE. Mature LY~ of both preparations migrated at 92 kDa in this system (data not shown).

In the reticulocyte lysate system, unglycosylated /3 is ex- pected to have a M, of -35,000 (Shull et al., 1986a). Our results are in accord with these estimates, implying that the 34-kDa product (obtained in the absence of microsomes) is full length (cf. Fig. 2, lane 6).

The apparent M, values of the N translation products were unchanged by the addition of dog pancreatic microsomes. Addition of microsomes, however, decreased the yield of “S- labeled a subunits (Fig. 2, cf. lanes 4 and 5). Addition of microsomes to the rabbit reticulocyte lysates incubated with

-+-+-+ 1234567

kDo

200-

1 kDa - 92-

I

+ a(921

69- -

df- - I.

* * w/3(40)

-iL

+f3(37) +pc351 +3(34)

30- -

14-

FIG. 2. Translation of cRNA, and cRNAB in rabbit reticu- locyte lysates. cRNA,, and cRNA, were translated in rabbit reticu- locyte lysates charged with I-[““Slmethionine, with (+) or without (-) added dog pancreatic microsomes. The entire mixtures were analyzed by 10% SDS-PAGE. Lane I, “C molecular weight standards (all methylated: myosin, 200,000; phosphorylase b, 92,500; bovine serum albumin, 69,000; ovalbumin, 46,000; carbonic anhydrase, 30,000; lysozyme, 14,300). Lanes 2 and 3, controls (no cRNA added). Lane 3, cRNA,, products in t.he absence of microsomes. Lane 5, cRNA,, products in the presence of microsomes. Lane 6, cRNAi, products in the absence of microsomes. Lane 7, cRNA,, products in the presence of microsomes.

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Transcription and Translation of Na, K-A TPase 10831

cRNAp gave rise to three more polypeptides with M, of 35,000, 37,000, and 40,000, respectively (Fig. 2, lane 7). The M, = 35,000, 37,000, and 40,000 products were likely the result of core glycosylation (Hubbard and Ivatt, 1981; Fambrough, 1983; Sweadner and Gilkeson, 1985). This inference was tested by treatment with endoglycosidase H and lectin binding studies (see below).

Inmunopreclpitation-Immunoprecipitation was used to verify the identity of the synthesized peptides. The LY products, including the lower molecular weight products, as well as the /3 products, obtained in the presence of pancreatic micro- somes, were precipitated with rabbit anti-lamb Na,K-ATPase (80g8) antiserum (Fig. 3, lanes 3,4,6, and 7). The specificity of immunoprecipitation of cy was verified by immunocompetition studies using purified undenatured lamb kidney Na,K-ATP- ase (cf. lanes 6 and 9, Fig. 3). In contrast, undenatured lamb kidney Na,K-ATPase had no effect on immunoprecipitation of the cRNA~ translation products (Fig. 3, compare lanes 7 and 10). Immunoprecipitation of p by polyclonal anti-holo- enzyme (809*) and by anti-@ (/3-Gly-2J antisera, however, was inhibited by preincubation with SDS-denatured lamb kidney Na,K-ATPase holoenzyme (data not shown). These results confirm the authenticity of the cRNA, and cRNA, products.

Membrane Integration-To differentiate between mem- brane-integrated and peripherally associated peptides, the translation products were alkali-extracted before centrifuga- tion and immunoprecipitation. To distinguish between co- translational and post-translational integration, dog pan- creatic microsomes were added to the translation mix either at the start of translation or 1 h after initiation of translation. In the latter case, the lysate-microsome mixture was further incubated for another 1 h at 30 “C before centrifugation.

In vitro translation of cRNAB in the presence of dog pan- creatic microsomes yielded a 34-kDa peptide that was re-

+Ab+ -Ab +A b holoenzvme

kDo -

zoo-

FIG. 3. Specificity of immunoprecipitation of Q and ,9 poly- DeDtideS. L-I”“SlMethionine-labeled Na.K-ATPase (Y and B subunits were synthesized in rabbit reticulocyte lysates, in the presence of dog pancreatic microsomes. Polyclonal rabbit anti-lamb Na,K-ATPase (anti-holoenzyme) antibodies (8098 Ab) were used to confirm the identity of the products by immunoprecipitation, as analyzed by 10% SDS-PAGE. Lane 1, “C-labeled molecular weight standards (see legend of Fig. 2). Lane 2, products obtained with no added cRNAs (control). Lane 3, yield of 01 polypeptide from the entire mixture. Lane 4, yield of fl polypeptide from the entire mixture. Lane 5, immuno- precipitates of the control incubation (compare to lane 2). Lane 6, immunoprecipitates of o( polypeptide (compare to lane 3). Lane 7, immunoprecipitates of p polypeptide (compare to lane 4). Lane 8, immunocompetition with purified lamb kidney holoenzyme of the controls, i.e. no added cRNAs (compare to lane 5). Lane 9, immuno- competition (with holoenzyme) of the immunoprecipitated 01 polypep- tide (compare to lane 6). Lane 10, immunocompetition (with holoen- zyme) of the immunoprecipitated p polypeptide (compare to he 7).

covered from both the supernatant and pellet fractions (Fig. 4, lanes 1 and 2), whereas the 35kDa, 37-kDa, and 40-kDa peptide products were present only in the pellet fraction, even after alkali extraction (Fig. 4, lanes 2 and 4). The yield of the 35-kDa, 37-kDa, and 40-kDa peptides was considerably greater when dog pancreatic microsomes were present at the start of translation (Fig. 4, lanes 2 and 4) than when added after completion of translation (Fig. 4, lanes 6 and 8). The traces of 35-40-kDa products obtained on adding microsomes post-translationally was eliminated by preincubating the translation mixture containing soluble p chains with ribonu- clease A (10 pg/32 ~1) and/or cycloheximide (1 mM) for 15 min, before addition of dog pancreatic microsomes (data not shown). These results imply that p is co-translationally inte- grated.

With cRNA,,, the in vitro synthesized products had nominal molecular weights of 140-kDa, 92-kDa, 82-kDa, and 76-kDa (Fig. 5, lane 2). The immunoprecipitated LY products were approximately equally partitioned between the supernatant and pellet fractions (Fig. 5, lanes 3 and 4). The relative amounts of (Y recovered from supernatant and precipitate fractions, however, varied with different batches of rabbit reticulocyte lysates and dog pancreatic microsomes. The su- pernatantipellet ratio varied from 0.25 to 0.75. The (Y poly- peptides in the pellet resisted extraction by alkali (Fig. 5, lanes 4 and 6). When the microsomes were added to reticulo- cyte lysates after a l-h preincubation with cRNA,, the pep- tides were found only in the supernatant fraction (Fig. 5, lanes 7 and 8). These results yield no evidence for post-translational insertion of LY subunits into microsomal membranes.

Analysis of Integration by Trypsinolysis-Treatment of mi- crosome-associated (Y with trypsin eliminated all of the high molecular weight products (i.e. 92,000, 82,000, and 76,000) and generated two new products at 29,000 and 25,000 (Fig. 6, lanes 2-5 and 12). Addition of Triton X-100 rendered only the resistant fragment at 29 kDa susceptible to trypsin hy- drolysis (Fig. 6, lane 7). These results imply that a segment of newly synthesized (Y polypeptide was inserted into endo- plasmic reticulum membranes. Treatment of membrane-as-

I2345678 *

46-

* f&e --

30-

FIG. 4. Membrane integration of @ polypeptide. cRNAd was translated in rabbit reticulocyte lysates in the presence of L-[%I methionine. Dog pancreatic microsomes were added either at the start of translation (lanes 1-4) or after the completion of translation (lanes 5-B), and then the incubation was continued for an additional 1 h at 30 “C. The lysates were then either added to a neutral sucrose solution or alkali-extracted and fractionated by centrifugation. S, P, SOH, and PO” represent the supernatant and pellet fractions after dilution in sucrose buffer or alkali extraction, respectively. All products were immunoprecipitated with 8098 Ab and analyzed by 10% SDS-PAGE. The molecular weight calibrations are indicated on the left-hand margin. Lane I, @ from the neutral supernatant fraction (co-transla- tional addition of microsomes). Lane 2, 6 from the neutral pellet fraction (co-translational). Lane 3, alkali-extracted p from the super- natant fraction (co-translational). Lane 4, alkali-extracted p from the pellet fraction (co-translational). Lane 5, p from the supernatant fraction (post-translational addition of microsomes). Lane 6, 0 from the neutral pellet fraction (post-translational). Lane 7, alkali-ex- tracted fl from the supernatant fraction (post-translational). Lane 8, alkali-extracted p from the pellet fraction (post-translational).

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10832 Transcription and Translation of Na,K-ATPase

46. m

14-N w - c - Total s P s, PO” s P

FIG. 5. Membrane integration of a polypeptide. cRNA,, was translated in rabbit reticulocyte lysates in the presence of L-[""S] methionine. Dog pancreatic microsomes were added either at the start of translation (lanes 3-6) or after completion of translation (lanes 7 and 8), and incubation was continued for an additional 1 h at 30 “C. The lysates were either diluted with a neutral sucrose buffer or alkali- extracted and fractionated by centrifugation. S, P, S~H, and P,,H are defined in the legend of Fig. 4. All products were immunoprecipitated with 809, Ab and analyzed by 10% SDS-PAGE. Lane I, “C molecular weight standards (see legend of Fig. 2). Lane 2, (Y from the complete mixture (unfractionated). Lane 3, N from the supernatant fraction (co-translational addition of microsomes). Lane 4, (Y from the pellet fraction (co-translational). Lane 5, alkali-extracted cy from the super- natant fraction (co-translational). Lane 6, alkali-extracted (Y from the pellet fraction (co-translational). Lane 7, (Y from the supernatant fraction (post-translational addition of microsomes). Lane 8, LY from the pellet fraction (post-translational). The 92-kDa product corre- sponds to full length a.

I234567 8 9 IO II 12 13 kDa

-x-d-- 0 IO’ 20’ 40’ 60’ 60’T 0 IO’ 20’ 46 60’ 6O’T

FIG. 6. Trypsin digestion of a and fl polypeptides. cRNA., and cRNA,, were translated in rabbit reticulocyte lysates charged with L-[““Slmethionine and in the presence of pancreatic microsomes. After completion of translation (1 h), tetracaine hydrochloride was added to inhibit signal protease, and trypsinolysis was carried out for the indicated time periods. Proteolysis carried out for 60 min in the presence of TritonX-100 (0.5%) is denoted by 60’2’. The products were analvzed bv 10% SDS-PAGE. Lane 1. W-labeled molecular weight standards (see Fig. 2). Lane 2, untreated a from the total incubation mixture. Lanes 3-5, (Y from the total mixture after prote- olysis for 10, 20, and 40 min, respectively. Lane 12, a from the total mixture after 60 min of proteolysis. Lane 7, N from the total mixture after proteolysis for 60 min in the presence of Triton X-100. Lane 8, untreated 6 from the total mixture. Lanes 9-11, 6 from the total mixture after proteolysis for 10, 20, and 40 min, respectively. Lane 6, 6 from the total mixture after 60 min of proteolysis. Lane 13, 6 from the total mixture after proteolysis for 60 min in the presence of Triton x-100.

sociated @ with trypsin converted the prominent 40-kDa band to a 37.5kDa band (Fig. 6, lanes 6 and 8-11). When trypsi- nolysis was carried out in the presence of Triton X-100, all of the ,6 products were eliminated (Fig. 6, lane 13).

Glycosylation of Membrane-associated fl Polypeptides-Ad- dition of endoglycosidase H to extracted membrane-associ- ated p products eliminated the 35-40-kDa products and

yielded 34-kDa ,6 polypeptides, indistinguishable from those obtained in translations without microsomes (Fig. 7). Further evidence of glycosylation of the fi subunit was demonstrated by its ability to bind to lectin. a polypeptides exhibited minimal binding to concanavalin A, and these small amounts were not elutable with 2 M mannose (Fig. 8, lanes 2-5). In contrast, /3 (i.e. 40 kDa and 35 kDa) did bind to concanavalin A and was specifically eluted with 2 M mannose (Fig. 8, lanes 6-9).

The Potential Dependence of o( and p Subunit Insertion on SRP Receptor-Pretreatment of the pancreatic microsomes with NEM inhibited glycosylation of the membrane-associ- ated p polypeptides (Fig. 9). Most of the fl polypeptide (34 kDa) was in the supernatant fraction, and no glycosylated forms were in the pellet (data not shown).

Similarly, (Y was virtually absent from the pellets of the reticulocyte/NEM-pretreated dog pancreatic microsome mix- ture (Fig. 10). The yield of (Y chains in the supernatant of the mixtures containing NEM-pretreated microsomes was about

kD0 - I 2 3 4 5 6 46-

*

14-b 1, - - -++ +

+E +E

FIG. 7. Endoglycosidase H treatment of j3. cRNAd was trans- lated in a rabbit reticulocyte lysate charged with L-[S”S]methionine in the absence (-) (lanes 1-3) or presence (+) (lanes 4-6) of dog pancreatic microsomes. All products were immunoprecipitated with 809a Ab and solubilized by boiling for 5 min with 1% SDS. Some fractions were then treated with endoglycosidase H (+I?). The molec- ular weight calibrations are indicated on the far left. Lane 1, untreated, unextracted (no SDS) 0. Lane 2, untreated, 6 extracted with SDS. Lane 3, SDS-extracted b treated with endoglycosidase H (+E). Lane 4, untreated, unextracted (no SDS) 6. Lane 5, untreated, 6 extracted with SDS. Lane 6, 6 extracted with SDS and treated with endogly- cosidase H (+E).

’ 2 345 kDa -

-“- 200-

14-A

FIG. 8. Binding of a and j3 polypeptides to concanavalin A. cRNA., and cRNA,$ were translated in rabbit reticulocyte lysates charged with L-[‘“Slmethionine in the presence of dog pancreatic microsomes. After completion of translation, SDS extracts were chro- matographed on concanavalin A columns and eluted twice with 2 M mannose. The products bound to concanavalin A after elution, and the concanavalin A eluates were analyzed by 10% SDS-PAGE. Lane I, ?-labeled molecular weight standards (see legend to Fig. 2). Lane 2, LY extracted from the entire lysate-microsome mixture. Lane 3, (Y adsorbed to concanavalin A after mannose elutions. Lane 4, cy in the first 2 M mannose eluate. Lane 5, cy in the second 2 M mannose ehrate. Lane 6, 6 extracted from the entire lysate-microsome mixture. Lane 7, 6 adsorbed to concanavalin A after mannose elutions. Lane 8, p in the first 2 M mannose eluate. Lane 9, 6 in the second 2 M mannose eluate.

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Transcription and Translation of Na,K-ATPase 10833

cot , 2 3 4 46- .-

*r

FIG. 9. Effect of pretreatment of dog pancreatic microsomes with N-ethylmaleimide on glycosylation of 0. cRNAd was trans- lated in rabbit reticulocyte lysates charged with L-[““S]methionine in the absence of added microsomes or in the presence of untreated microsomes or NEM-treated microsomes. The @ products were ana- lyzed by 10% SDS-PAGE. Lane I, “‘C molecular weight standards (see legend to Fig. 2). Lane 2, /3 synthesized in the absence of microsomes (total mixture). Lane 3, fl synthesized in the presence of untreated microsomes (total mixture). Lane 4, 0 synthesized in the presence of NEM pretreated microsomes (total mixture).

I234 ‘1 m@nn gcJ

- a -200

re& ,s P, ,s P, -NEM tNEM

FIG. 10. Effect of pretreatment of dog pancreatic micro- somes with IV-ethylmaleimide on membrane integration of CX. cRNA,, was translated in rabbit reticulocyte lysates charged with L- [““S]methionine in the presence of untreated microsomes or NEM- treated microsomes. The (Y products were added to a neutral sucrose solution and fractionated by centrifugation. All products were im- munoprecipitated with 8098 Ab and analyzed by 10% SDS-PAGE. Lane I, (Y synthesized in the presence of untreated microsomes (supernatant fraction). Lane 2, (Y synthesized in the presence of untreated microsomes (precipitate fraction). Lane 3, (Y synthesized in the presence of NEM-pretreated microsomes (supernatant fraction). Lane 4, (Y synthesized in the presence of NEM-pretreated microsomes (precipitate fraction).

the same as that of the combined supernatant and pellet fractions of the untreated controls.

DISCUSSION

The present experiments were designed to examine various features of translation of mRNA, and mRNA, of Na,K- ATPase, in a cell-free system, without the complications of simultaneous translation of a multitude of other messages. Translation of cRNA,,, in a reticulocyte lysate yielded multiple peptide bands, with a prominent band at a nominal 92 kDa. Similarly, products with nominal M, of 70,000-135,000 were obtained in a variety of other systems with native mRNA, and synthetic cRNA,, preparations (Hiatt et al., 1984; Geering et al., 1985; Tamkun and Fambrough, 1986; Akayama et al., 1986; Homareda et al., 1988). The identity of the LY products in our preparations was confirmed by specific immunoprecip- itation with anti-Na,K-ATPase antisera.

The multiple a products could result from proteolytic cleav- ages of full length peptide but addition of a series of protease inhibitors to the translational mixture including PMSF, benz- amidine, leupeptin, and pepstatin had no effect on the yield of a products (data not shown). Alternatively, use of more than one initiation site or premature termination might have truncated the products at either the NH, terminus or COOH

terminus perhaps because of secondary structure formation in cRNA,, (Hiatt et al., 1984). In the present studies, however, the yield of multiple a products was unaffected by denatura- tion of the lamb cRNA,, with methyl mercury hydroxide or with heating (data not shown). Earlier studies indicated that mRNA,, does not code for a cleavable signal sequence and the mature polypeptide is nonglycosylated (Shull et al., 1985; Hiatt et al., 1984). As expected, the LY products derived from cRNA,, were the same size when synthesized in the presence or absence of dog pancreatic microsomes. The peptide band at a nominal 140 kDa (Fig. 3) is probably an a2 dimer. With poly(A+) translation, Hiatt et al. (1984) obtained a product at 135 kDa which was identified as an a:@ dimer.

In the presence of microsomes, translation of cRNAp yielded increments in M, (35,000, 37,000, and 40,000) and no decrement indicative of a cleavable sequence, as predicted by sequence analysis (Shull et al., 1986a). These results are in accord with earlier studies on the biosynthesis of /? (Fam- brough, 1983; Geering et al., 1985; Tamkun and Fambrough, 1986; Gilmore-Hebert et al., 1988; Homareda et al., 1988). The 35-40-kDa products apparently are core glycosylated via ad- dition of N-linked high mannose chains, in that these prod- ucts exhibited mannose-sensitive binding to concanavalin A and were cleaved by incubation with endoglycosidase H to the protein core (Tarentino and Maley, 1974; Shull et al., 1986a; Wolitzky and Fambrough, 1986; Akayama et al., 1986). In addition, Kawakami and Nagano (1988) using deletion mu- tants reported that the single membrane anchor domain of the human fi subunit was required for membrane insertion, and that integration was SRP-dependent, based on inhibition with a polyvalent anti-SRP antibody.

Stable, alkali-resistant association of the cy or p products with the microsomal membranes was obtained only co-trans- lationally, in that post-translational mixing experiments yielded no membrane-associated products in either case. In the reticulocyte lysate-pancreatic microsome system, (Y was partitioned -50:50 and p -20:80, between the soluble and particulate phases. Of the membrane-associated (Y, 15 to 50% of the mass was resistant to alkali extraction, compared to -90% of alkali-resistant membrane-bound /?. These results imply somewhat more efficient membrane integration of /3, as compared to CL They also raise the possibility that some other factor may be required for efficient integration of a, as pro- posed by Hiatt et al. (1984).

The possibility that membrane association of a may be dependent on prior or simultaneous uptake of /3 was assessed in combination experiments. Addition of cRNAd to the retic- ulocyte lysate-microsome mixture 20 min before the addition of cRNA,, reduced the yield of membrane-associated (Y by -75% (data not shown). Simultaneous addition of cRNA, and cRNA, gave no enhancement of the yield of either membrane- associated cy or fi (data not shown). Thus, no evidence was adduced for /3 acting as a “receptor” for membrane integration of a!.

Proteolysis of membrane-associated LY yielded a small re- sistant fragment (29 kDa). In contrast, -90% of the mass of membrane-associated @ was trypsin-resistant. Our results are in accord with those of Geering et al. (1985) who also found that -35% of the mass of membrane-associated a (poly(A+) RNA of amphibian origin) was trypsin-resistant, whereas the core-glycosylated @ product was -95% resistant. Newly syn- thesized membrane-associated cy subunits were also found to be extensively degraded by trypsin in the studies of Hiatt et al. (1984) and Homareda et al. (1989). Almost all of core glycosylated /3 was trypsin-resistant, implying close to com- plete translocation into the cisternae, as reported for the

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10834 Transcription and Translation of Na,K-ATPase

human /3 subunit by Kawakami and Nagano (1988). The present findings, as well as those of Geering et al.

(1985), Homareda et al. (1988, 1989), Kawakami and Nagano (1988), and Gilmore-Hebert et al. (1988) support the inference of co-translational membrane integration of both CY and /3 subunits. These results imply that mRNA, and mRNAs should be recoverable primarily from membrane-bound poly- somes. In the studies reported to date, however, mRNA, was recovered from free polysomes by Hiatt et al. (1984) and from membrane-bound polysomes by Fisher et al. (1984). Fisher et al. (1984) also reported the recovery of mRNAp from free polysomes. These conflicting results remain to be resolved by future studies.

To evaluate the possible participation of the SRP receptor in membrane integration of CY and ,f3 subunits, translation was attempted in the presence of pancreatic microsomes pre- treated with NEM (Holland and Drickamer, 1986), after which neither subunit exhibited uptake into the endoplasmic reticulum. Homareda et al. (1989) also found that pretreat- ment of microsomes with NEM inhibited membrane associa- tion of the human cy subunit. These results implicate a protein, possibly the SRP receptor, in the uptake of the (Y subunit.

The absence of cleavable signal sequences indicates that both Q and p subunits belong to type II transmembrane proteins (Holland and Drickamer, 1986). Unlike type I, the amino termini of type II transmembrane proteins are cyto- plasmic (Steiner et al., 1980; Engelman and Steitz, 1981). The adjacent membrane-spanning hydrophobic sequences are in- serted into the membranes in an SRP-dependent manner involving the same mechanism as those of type I transmem- brane proteins. The internal signal sequence in the primary structure of the polypeptide determines its orientation in the lipid bilayer (Holland and Drickamer, 1986). Based on various deletion mutants of the cDNA encoding (Y and @ subunits, it was proposed that the first four of the NHz-terminal six or seven transmembrane segments encode the membrane inser- tion signals of the LY subunit and that at the minimum a 16- amino acid stretch in the transmembrane segment of the /3 subunit is required for its insertion (Kawakami and Nagano, 1988; Homareda et al., 1989).

Now that (Y and 0 subunits can be synthesized in vitro, their assembly and subsequent functional activity may be amenable to study. Although no enzymatic activity has thus far been demonstrated in membranes containing co-translated (Y and p subunits, functional Na,K-ATPase was detected in oocytes injected with synthetic (Y and @ subunit-mRNAs derived from cloned Torpedo culifornicu cDNAs (Noguchi et al., 1987; Gil- more-Hebert et al., 1988).

Acknowledgments-It is a pleasure to thank Dr. Kurt Drickamer for his advice and criticisms. We are indebted to Drs. Gary E. Shull, Jerry B. Lingrel, and W. James Ball for providing cDNAs and antibodies. Vincent Wu’s skilled technical assistance was most help- ful.

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Transcription and Translation of Na,K-ATPase

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E Cayanis, H Bayley and I S EdelmancDNAs.

Cell-free transcription and translation of Na,K-ATPase alpha and beta subunit

1990, 265:10829-10835.J. Biol. Chem. 

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