©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Four Hydrophobic Segments in the NH-terminal Third (H1H4) of Na,K-ATPase Subunit Alternately Initiate and Halt Membrane Translocation of the Newly Synthesized Polypeptide (*)

Yiheng Xie (§) , Takashi Morimoto (¶)

From the (1) Department of Cell Biology and Kaplan Cancer Center, New York University School of Medicine, New York, New York 10016

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Transmembrane disposition of the NH-terminal third of the Na,K-ATPase subunit was studied using an experimental approach that involved in vitro endoplasmic reticulum membrane insertion of chimeras. These chimeras consisted of four truncated amino-terminal segments of the subunit linked at amino acid residues 126, 179, 313, and 439 to chloramphenicol acetyltransferase (CAT), a reporter protein, that contains a consensus sequence for N-linked glycosylation. The fusion sites were located after one of the four hydrophobic segments (H1-H4). The results showed that the chimeras in which the subunit was truncated at positions 126 and 313 were glycosylated, and the glycosylated peptides were protected by membranes from proteolysis. However, the other two chimeras were not glycosylated and the inserted peptides were digested by protease into fragments which did not immunoprecipitate with anti-CAT. These results clearly demonstrate that hydrophobic segments H1 and H3 function as signal/anchor type II, and H2 and H4 function as halt transfer signals. Furthermore, membrane insertion of the NH-terminal third of Na,K-ATPase subunit is achieved by a series of alternate signal/anchor type II and halt transfer sequences.


INTRODUCTION

Na,K-ATPase of animal cells is an integral plasma membrane protein which plays an important role in maintaining the characteristic ionic composition of cytoplasm and in generating electro-chemical gradients necessary for normal cell function (see reviews by Jorgensen and Andersen(1988) and Skou and Esmann(1992)). The enzyme consists of two noncovalently linked subunits in an equimolar ratio (Brotherus et al., 1983): a larger nonglycosylated subunit (about 100 kDa), and a glycosylated subunit (about 55 kDa) (Glynn, 1985; Jorgensen, 1982), both of which are transmembrane proteins (see review by Ovchinnikov(1987)).

The complete amino acid sequence of the Na,K-ATPase subunit has been deduced from the nucleotide sequence of cDNA cloned from various sources (Shull et al., 1986; Kawakami et al., 1985 and 1986; Noguchi et al., 1986; Hara et al., 1987). The hydropathy analysis has shown that the subunit has eight to ten hydrophobic segments. Four segments (H1-H4) are present in the NH-terminal third, and the rest of four to six segments are present in the COOH-terminal third. Orientation of the first four hydrophobic segments in the lipid bilayer has been investigated by a variety of approaches, including immunochemical methods (Farley et al., 1986; Ball and Loftice, 1987; Felsenfeld and Sweadner, 1988; Kano et al., 1990; Arystarkhova et al., 1992; Mohraz et al., 1994), proteolytic digestion (Ovchinnikov et al., 1987), site-specific labeling (Jorgensen et al., 1982; Nicholas, 1984; Capasso et al., 1992; see also review by Mercer(1993)). These results demonstrated that the NH terminus is exposed on the cytoplasmic side of the membrane and that the predicted four hydrophobic segments appear to be transmembrane domain. Recently Homareda et al.(1989) constructed truncated subunits containing two adjacent hydrophobic segments (H1 and H2, H3 and H4, or H5 and H6) and examined if these truncated peptides were inserted into dog pancreas microsomal membranes in N-ethylmaleimide-sensitive fashion using a rabbit reticulocyte lysate translation system. Their results have shown that at least two insertion signals are present within the four hydrophobic segments in the NH-terminal third.

In the present paper, we describe the studies on the transmembrane disposition of the NH-terminal third of subunit using an experimental strategy based on the topogenic properties of the four hydrophobic segments H1-H4. In this approach, a reporter protein chloramphenicol acetyltransferase (CAT)() that has a consensus N-linked glycosylation site is linked sequentially in frame after each hydrophobic segment in four truncated subunits of different lengths. CAT is chosen since it is a cytosolic protein and does not contain either insertion or halt transfer sequences. It can therefore be used as a faithful COOH-terminal reporter protein (Lipp and Dobberstein, 1986, 1988). The chimeras are designed such that topogenic properties of hydrophobic segments can be examined sequentially in the native context. Signal/anchor and halt transfer sequences are identified by characterization of their COOH-terminal flanking sequence with respect to the membrane. Two properties, occurrence of N-linked glycosylation and membrane protection of the reporter protein from proteolysis, are used as the marker for the luminal disposition. The results, therefore, will provide information on both the orientation (with respect to the membrane) and the function (an insertion signal, a halt transfer signal or topogenically inactive element) of each hydrophobic segment in the native context.


EXPERIMENTAL PROCEDURES

Materials

Restriction enzymes, SP6 RNA polymerase, DNA polymerase I (Klenow enzyme), T4 DNA ligase, T4 polynucleotide kinase, RNasin, Taq DNA polymerase, m7G(5`)ppp(5`)G, and calf intestinal alkaline phosphatase were obtained from either Boehringer Mannheim or Life Technologies, Inc. Tetracaine, phenylmethylsulfonyl fluoride, trypsin, chymotrypsin, and Trasylol (aprotinin) were obtained from Sigma. The Geneclean kit was purchased from BIO 101. Reticulocyte lysate and dog pancreas microsomal membranes were obtained from Promega Corp. (Madison, WI). [S]Methionine was obtained from DuPont NEN. Rabbit antibody against CAT was obtained from 5 Prime 3 Prime Inc. (Boulder, CO).

Plasmids and Construction of Mutants

Plasmids pGEM-3Z and pCAT basic were obtained from Promega Corp., and pT7/T3 19R was obtained from Life Technologies, Inc. Okayama-Berg vector containing an entire coding region of rat brain Na,K-ATPase -1 subunit was supplied by Dr. Y. Hara (Tokyo Dental Medical School, Tokyo).

All cDNA constructs (pN-1, pN-2, pN-3, and pN-4) encoding the fusion proteins of the truncated Na,K-ATPase subunit and CAT as a reporter protein were derived from two plasmids: pF1 in which the entire coding region of -1 subunit was engineered into a plasmid pT7/T3 19R, and pCAT, in which the entire coding region of CAT was engineered into a plasmid pGEM-3Z. cDNA constructs pX-1, pX-2, pX-3, and pX-4 were constructed from an in vivo and in vitro expression vector containing rat growth hormone cDNA (pSG5rGH) and one of the pN-1, pN-2, pN-3, and pN-4.

pF1

The -1 subunit cDNA, engineered from Okayama-Berg vector into pT7/T3 19R vector, contained about 200 nucleotides in 5` end-noncoding region. When the mRNA from the cDNA was translated in a rabbit reticulocyte lysate system the translation efficiency was very poor probably due to the long stretch of GC pairs preceding the initiation codon. To improve the in vitro translation efficiency, a new 45-mer oligonucleotide, AGCTTGATATCGATAACTGAATAGCTGCAGGACGATCGCCATGGG, was designed according to Kozak's rule (Kozak, 1987) and engineered just before the initiator codon of the subunit by ligating the annealed oligomers, the NcoI/SacI fragment from plasmid Okayama-Berg 1 and the plasmid pT7/T3 19R cut with HindIII and NcoI.

pCAT

The entire coding region of the CAT gene obtained by cutting a plasmid pCAT basic with HindIII and NlaIV was ligated into the plasmid pGEM-3Z cut with HindIII and HincII.

pN-1

The 404-base pair fragment obtained by cutting a plasmid pF I with PstI and RsaI was ligated into the plasmid pCAT that had been cut with PstI and HincII.

pN-2

A plasmid pF 1 was linearized by NspV digestion, and its protruding ends were filled in with Klenow enzyme. The linearized, blunt-ended pF 1 was then ligated to 8-mer SalI linkers, and the ligated sample was digested with PstI and SalI. A 535-base pair PstI/SalI fragment thus obtained was inserted into the plasmid pCAT that had been cut with PstI and SalI.

pN-3

A plasmid pF 1 was digested with XhoI, and the protruding ends were filled in with Klenow enzyme. After digestion with PstI, an approximately 0.97-kilobase pair fragment was isolated and inserted into the plasmid pCAT that had been cut with PstI and HincII.

pN-4

A plasmid pF 1 was linearized with AflII, and its protruding ends were filled in with Klenow enzyme. After digestion with PstI, a fragment of about 1.35 kilobase pair was isolated and inserted into the plasmid pCAT that had been cut with PstI and HincII.

pX-1, pX-2, pX-3, and pX-4

A fragment containing one of four hydrophobic segments (H1, H2, H3, or H4) and CAT was made by polymerase chain reaction using their corresponding linearized plasmid pN-1, pN-2, pN-3, or pN-4 as the template and two oligonucleotides described below. The polymerase chain reaction product was digested with NaeI (pX-1 and pX-2) or PmlI (pX-3 and pX-4) and BamHI and then ligated to the plasmid pSG5rGH that had been cut with Eco47III and BamHI. The NH-terminal side oligonucleotides (oligonucleotides 1, 2, 3, and 4) and the COOH-terminal side oligonucleotide (oligonucleotide C) are: oligonucleotide 1 for pX-1, ATTCCGGCAGCTGTT; oligonucleotide 2 for pX-2, GATGAGCTCTGCCGGCTGTACCTCGGGGTCGTGGC; oligonucleotide 3 for pX-3, GATGAGCTCGCACGTGTCATCCACCTCATCACGGG; oligonucleotide 4 for pX-4, TGATGAGCTCGCACGTGCTGTCATCTTCCTCATTGG; oligonucleotide C, CCATGGCTCGAGCTTAAG.

That all constructs encoding chimeras were ligated in frame has been confirmed by DNA sequencing, size of the primary translation products, and immunoreactivity of the primary translation products to anti-CAT antibody.

Dog Pancreas Microsomes (Referred to as ``Microsomes'' in This Paper Unless Specified)

Microsomes were prepared from canine pancreas as described by Walter and Blobel(1983). Occasionally microsomal membranes were obtained from Promega Corp.

In Vitro Transcription

The plasmids pN-1, pN-2, pN-3, and pN-4 and pX-1, pX-2, pX-3, and pX-4 were cut with SmaI and BamHI, respectively, and the linearized DNAs were purified with a Geneclean kit. A 45 µl of transcription reaction mixture contains 5 µg of linearized plasmid DNA, 80 mM HEPES-KOH (pH 7.5), 16 mM MgCl, 2 mM spermidine, 40 mM DTT, 1 unit/µl RNasin, 25 µg/ml bovine serum albumin, 0.5 µM cap analog (m7G(5`)ppp(5`)G), and 50 units of SP6 RNA polymerase (for pN-1, pN-2, pN-3, and pN-4) or T7 RNA polymerase (for pX-1, pX-2, pX-3, and pX-4). After the mixture was incubated for 10 min at 40 °C, 5 µl of nucleotide mixture (0.5 mM each of ATP, CTP, UTP, and GTP) was added and incubated again at 40 °C for 1 h. Then, in order to obtain the maximal amount of the transcripts, 50 units of RNA polymerase were added, and the reaction mixture was incubated at 40 °C for an additional hour. The transcripts were then purified with phenol/chloroform extraction, followed by ethanol precipitation, and the pellets were dissolved in 50 µl of diethyl pyrocarbonate-treated water.

In Vitro Translation

The translation reaction was carried out as described in the Promega's technical manual. Briefly, a 20-µl translation mixture contains 14 µl of reticulocyte lysate, 1.6 µl of 10 mCi/ml [S]methionine, 20 µM each of the other 19 amino acids, 20 units of RNasin, and 68 mM KOAc (endogenous amount of KOAc, 78 mM, contained in the lysate was not included). The reaction mixture was incubated at 30 °C for 90 min in the presence or absence of microsomal membranes (5 A units/ml of translation reaction).

Isolation of Microsomal Membranes from the Translation Mixture

Immediately after the incubation was over, the translation mixture was chilled on ice and diluted with high salt buffer (500 mM KOAc, 3 mM Mg(OAc), 50 mM triethanolamine (pH 7.5), and 1 mM DTT) to 100 µl. The diluted sample was incubated on ice for an additional 10 min and overlaid onto a 100-µl cushion containing 0.5 M sucrose, 500 mM KOAc, 50 mM triethanolamine, 3 mM Mg(OAc), and 1 mM DDT. The step gradient was centrifuged at 60,000 rpm for 150 min in the 100.3 rotor of the Beckman centrifuge TL-100. The top 125 µl was collected as the supernatant fraction, the rest of the sucrose cushion was discarded, and the pellet (microsomal fraction) was gently washed once with high salt buffer.

Removal of Globin from the Translation Reaction Mixture

The supernatant fractions (see the previous paragraph) or the translation products synthesized in the absence of membranes were mixed with 1.7 volume of saturated ammonium sulfate solution. Trasylol was added to a final concentration of 2 µg/ml. The mixture was incubated on ice for 30 min and centrifuged at 4 °C for 4 min. The resultant pellets were suspended in a small volume of 5% trichloroacetic acid and centrifuged at 4 °C for 4 min in an Eppendorf centrifuge. The pellets were suspended in a small volume of 0.1 M Tris-HCl (pH 9.2), 2% SDS, and 50 mM DTT. The suspension was incubated for 30 min at 55 °C and then analyzed in 12.5% SDS-PAGE.

Endoglycosidase H (Endo H) Treatment

The microsomal membranes isolated from the translation mixture as described earlier were dissolved in a 30 µl 1% SDS solution containing 50 mM DTT, incubated for 3 min in boiling water, followed by addition of an equal volume of 0.2 M sodium citrate buffer (pH 5.5) containing 0.2 µg/ml Trasylol. The samples were then equally divided into two parts: one part was supplemented with 3 µl of 1 mU/µl Endo H and to the other 3 µl of water was added. Both samples were incubated at 37 °C overnight. After addition of 33 µl of the sample loading buffer (0.1 M Tris-HCl pH 9.2, 50 mM DTT and 10% glycerol) the samples were incubated in boiling water for 3 min and analyzed in 12.5% SDS-PAGE.

Immunoprecipitation

Microsomal membranes isolated from the translation mixture were suspended in 40 µl of dilution buffer (2.5% Triton X-100, 190 mM NaCl, 6 mM EDTA, 50 mM Tris-HCl (pH 7.4)) containing 0.1 µg/ml Trasylol. 10 µl of 20% SDS solution was added, and the sample was incubated in boiling water for 4 min. After the sample was chilled to about 4 °C, 4 volumes of cold dilution buffer and then 4 µl of antiserum against CAT were added. The samples were processed as described by Anderson and Blobel(1983). For immunoprecipitation of the translation products synthesized in the absence of microsomal membranes, immunoprecipitation was conducted after the endogenous globins were removed by addition of saturated ammonium sulfate, as described before.

Protease Digestion

Translation mixture was diluted with 3 volumes of 50 mM Tris-HCl (pH 7.5), 100 mM NaCl, and incubated in the presence of 3 mM tetracaine hydrochloride at room temperature for 10 min to stabilize the microsomal membranes. Freshly prepared stock solutions of trypsin and chymotrypsin were added to a final concentration of 100 µg/ml each in the presence or absence of 1% Triton X-100. Proteolytic digestion was carried out at room temperature for 30 min and terminated by the addition of Trasylol (at a final concentration of 2 µg/ml) and phenylmethylsulfonyl fluoride (at a final concentration of 3 mM). The reaction mixture was then supplemented with KOAc to a final concentration of 400 mM and overlaid onto 100 µl of 0.5 M sucrose/0.5 M KOAc cushion. The step gradient was centrifuged at 60,000 rpm for 15 min in the 100.3 rotor of the Beckman centrifuge TL-100. The membrane pellets were washed once with 50 µl of high salt buffer solution and analyzed in SDS-PAGE after incubation in boiling water for 5 min in the presence of sample loading buffer.

RESULTS

As shown in the hydropathy plot (Fig. 1), the NH-terminal third of the Na,K-ATPase subunit contains four hydrophobic segments. They are located at residues Leu-Ile (H1), Leu-Gln (H2), Phe-Leu (H3), and Ala-Ala (H4). The topogenic nature of these hydrophobic segments was examined using in vitro insertion into endoplasmic reticulum (ER) of chimeras that were expressed by the cDNA constructs pN-1, pN-2, pN-3 and pN-4. These constructs contained one or more hydrophobic segments sequentially in their native context. As described in the Introduction, two criteria, occurrence of N-linked glycosylation of the reporter protein and membrane protection of the reporter protein from proteolysis, were used as markers for the luminal location of the ligation site.


Figure 1: Amino acid sequence (NH-terminal portion) deduced from the nucleotide sequence of rat brain Na,K-ATPase -1 cDNA and its hydropathy plot. Amino acids are numbered beginning at Gly of the mature protein and preceded by 5 amino acid residues present in the primary translation product (Hara et al., 1987). Hydrophobic segments (H1-H4) are boldface and are also indicated by a broken line. The sites at which truncated subunits are linked in frame with COOH-terminal reporter CAT protein at cDNA level are indicated by an upward arrow. Hydropathy plot was based on the Kyte and Doolittle scale. Hydrophobic regions are above the x axis and hydrophilic regions are below the axis.



Hydrophobic Segment H1 Functions as a Signal/Anchor Type II

When the mRNA transcribed from pN-1 was translated in a rabbit reticulocyte lysate in the absence of microsomal membranes, the immunoprecipitates with anti-CAT antibody contained a single band with molecular mass of 39 kDa, which corresponds to the size of the primary translation product of the fusion protein N-1 (Fig. 2, lane 1). Translation in the presence of microsomal membranes yielded two major proteins with molecular masses of about 42 and 39 kDa (Fig. 2, lane 2). This 39-kDa protein was slightly larger than the primary translation product in lane 1, which suggested that the 39-kDa protein was not the primary translation product but the glycosylated fusion protein N-1 that was initiated from an internal methionine, Met. This is supported by Endo H digestion results discussed below. The 42-kDa protein comprised about 60% of the total immunoprecipitates. The immunoprecipitates also contained a smaller protein (about 36 kDa) as a minor product. Since an internal methionine residue is located at the 32nd amino acid from the NH terminus (see Fig. 1), the minor protein may be the unglycosylated fusion protein N-1 that was initiated by the internal methionine. When the immunoprecipitates shown in lane 2 were digested with Endo H, the two major proteins shifted quantitatively from 42 and 39 kDa to 39 and 36 kDa, respectively (lane 3). This 36-kDa protein migrated exactly at the same speed as the minor protein in lane 2, strongly supporting that the 39-kDa protein in lane 2 was the glycosylated fusion protein N-2 initiated by Met Therefore, the glycosylated form comprised more than 80% of the newly synthesized fusion proteins. This high efficiency of translocation of the fusion protein N-1 initiated by the hydrophobic segment H1 allowed us to examine the topogenic properties of its downstream hydrophobic segments in an in vitro translation/translocation system.


Figure 2: In vitro membrane insertion of chimeras N-1, N-2, N-3, and N-4. Top, in vitro transcripts of pN-1, pN-2, pN-3, and pN-4 were translated in a reticulocyte lysate in the absence (lanes 1, 4, 6, and 9) and presence (lanes 2, 3, 5, 7, 8, and 10) of microsomes. After translation, all samples were subjected to immunoprecipitation with anti-CAT antibody, followed by SDS-PAGE analysis as described under ``Experimental Procedures.'' Mb and Endo H represent microsomes and endoglycosidase H, respectively. Open and solid circles indicate glycosylated and unglycosylated forms, respectively. Bottom, schematic drawing shows the orientation of the inserted chimeras, based on the above results. Solid hearts and diamonds indicate glycosylated forms and potential N-linked glycosylation sites, respectively. N and C represent NH and COOH termini, respectively.



To examine whether the hydrophobic segment H1 functions as an anchor signal, the fusion protein N-1, which was synthesized in the presence of microsomes, was digested with trypsin and chymotrypsin in the presence or absence of Triton X-100. The membrane-protected fragments were recovered by immunoprecipitation with anti-CAT antibody. In this experiment, if H1 functioned as an anchor signal, 95 NH-terminal flanking residues of H1 would remain untranslocated in the cytoplasmic side of the membrane and would be cleaved by proteases in the absence of Triton X-100. Therefore, the size of a peptide that is protected by membranes from proteolysis will be smaller by about 10 kDa than the control fusion protein N-1. As shown in Fig. 3, lane 2, one fragment whose molecular mass was about 30 kDa was recovered by immunoprecipitation when digested in the absence of Triton X-100. As no fragments were recovered upon digesting in the presence of Triton X-100 (Fig. 3, lane 3), it is reasonable to conclude that the fragment recovered was a membrane protected one. These results clearly demonstrated that the hydrophobic segment H1 functioned as an insertion signal as well as an anchor of the inserted signal sequence and also confirmed that the NH-terminal flanking region of the H1 is located in the cytoplasmic side of the membrane.


Figure 3: Sensitivity to proteolysis of the inserted chimeras N-1, N-2, N-3, and N-4. Top, mRNAs derived from pN-1, pN-2, pN-3, and pN-4 were translated in a reticulocyte lysate in the presence of microsomes. One-third of the translation mixture was used as a control (lanes 1, 4, 7, and 10), one-third was digested with trypsin and chymotrypsin in the absence of Triton X-100 (lanes 2, 5, 8, and 11), and one-third was digested with trypsin and chymotrypsin in the presence of Triton X-100 (lanes 3, 6, 9, and 12). All samples were subjected to immunoprecipitation with anti-CAT antibody, followed by SDS-PAGE analysis as described under ``Experimental Procedures.'' Mb, Trypsin, and Triton represent microsomes, trypsin and chymotrypsin, and Triton X-100, respectively. Open and solid circles indicate glycosylated and unglycosylated forms, respectively. Bottom, schematic drawing that shows the membrane orientation of the fragments protected by microsomal membranes from proteolysis. Solid hearts indicate glycosylated forms.



Hydrophobic Segment H2 Functions as a Halt Transfer Signal

Upon translation of the plasmid pN-2 transcript in a reticulocyte lysate without addition of microsomes, a major protein with molecular mass of about 51 kDa was recovered by immunoprecipitation with anti-CAT antibody (Fig. 2, lane 4). This size corresponds to that of the unglycosylated fusion protein N-2. A minor band (about 48 kDa) was also recovered. This protein was probably an unglycosylated fusion protein N-2 that was initiated by the internal Met. The molecular mass difference between the major and minor proteins was about 3 kDa, which corresponds to the size of the peptide between Met and Met. When the pN-2 transcripts were translated in the presence of microsomes, a 51-kDa protein was recovered as a major product (Fig. 2, lane 5). This product had the same apparent molecular mass as those obtained in the absence of microsomes (compare the sizes of the major bands in lanes 4 and 5), which strongly indicated that the fusion protein N-2 was not glycosylated. As predicted, the apparent molecular mass of this band did not shift down by Endo H treatment (the data are not shown here). Since the CAT protein was linked to an amino acid residue in the COOH-terminal flanking region of H2, the result showed that the translocation which was initiated by H1 was halted by H2, and consequently the CAT protein remained in the cytoplasmic side of membranes as illustrated in Fig. 2, bottom. These results were further supported by proteolysis experiments. Fig. 3 , lane 5, showed that no membrane-protected fragments were recovered by immunoprecipitation upon digestion with proteases.

The Third Hydrophobic Segment H3 Functions as a Signal/Anchor Type II

The immunoprecipitates from the translation of pN-3 transcript in the absence of microsomes contained a major band with molecular mass of about 59 kDa (Fig. 2, lane 6), which is the size of an unglycosylated fusion protein N-3. A minor band (about 56 kDa) might be an unglycosylated fusion protein that was initiated by an internal methionine. Translation in the presence of microsomes yielded an additional band with molecular mass of about 62 kDa (Fig. 2, lane 7). The larger band, however, shifted its molecular mass to 59 kDa by Endo H treatment (Fig. 2, lane 8), which confirmed that the shift was due to deglycosylation of the N-linked sugar moiety. Since hydrophobic segment H3 was linked to an amino acid residue in the COOH-terminal flanking region of H3, the results demonstrated that the second round of translocation of the N-3 peptide was initiated by H3, and the following CAT protein was translocated into the ER lumen where it was glycosylated.

To confirm if the hydrophobic segment H3 functions also as an anchor signal, the translation mixture performed in the presence of microsomes was digested with trypsin and chymotrypsin, and the digested sample was subjected to immunoprecipitation with anti-CAT antibody. As predicted, only one fragment (about 29 kDa) was recovered by the immunoprecipitation (Fig. 3, lane 8), and no fragments were recovered upon proteolysis in the presence of Triton X-100 (Fig. 3, lane 9). This verified that the 29-kDa band was the membrane-protected fragment, and the results confirmed that the hydrophobic segment H3 functioned as a signal/anchor type II.

The Fourth Hydrophobic Segment H4 Functions as a Halt Transfer Signal

Translation of the plasmid pN-4 transcript carried out in a reticulocyte lysate without addition of microsomal membranes produced a major band with molecular mass of about 70 kDa (Fig. 2, lane 9), which corresponds to the unglycosylated form of the fusion protein N-4. Translation performed in the presence of microsomes produced also a 70-kDa protein (Fig. 2, lane 10). This result indicated that the H4 halted the translocation of its downstream peptide containing CAT protein, and therefore, the CAT protein remained in the cytoplasmic side of the membranes.

To ascertain that the CAT protein remained untranslocated, the translation mixture of the pN-4 transcript, carried out in the presence of microsomes, was digested with trypsin and chymotrypsin, and the digested sample was subjected to immunoprecipitation with anti-CAT antibody. No fragments were recovered (Fig. 3, lane 11). Thus, protease digestion experiments demonstrated that the CAT protein remained in the cytoplasmic side of membranes and was digested by exogenously added proteases.

Hydrophobic Segments H1 and H3 Are Also Capable of Halting Translocation of Their Downstream Peptide

Fig. 2 and Fig. 3have shown that hydrophobic segments H2 and H4 function as halt transfer signal in the native context. In order to examine whether the topogenic nature of H2 and H4 is intrinsic and whether H1 and H3 are also capable of halting translocation, we chose a rat growth hormone signal sequence and constructed plasmids pX-1, pX-2, pX-3, and pX-4. These plasmids encoded fusion proteins consisting of the NH-terminal 66 amino acid residues (including a cleavable signal sequence) of rat growth hormone and one of the hydrophobic segments (H1, H2, H3, and H4) linked at its COOH-terminal flanking region to the CAT protein. As a control, plasmid pX-c was constructed. This plasmid encoded a chimera consisting of NH-terminal 66 amino acid residues of rat growth hormone and CAT protein only. In this experiment, the topogenic nature of each hydrophobic segment was characterized by occurrence of N-linked glycosylation of the reporter protein and complete protection by membranes from proteolysis, as shown schematically in Fig. 4B.


Figure 4: A, topogenic properties of the four hydrophobic segments examined individually (all function as halt transfer signals). mRNAs derived from the plasmids pX-c, pX-1, pX-2, pX-3, and pX-4 were translated in a reticulocyte lysate in the absence (lane 1) or presence (lanes 2-5) of microsomes. The latter translation mixtures were divided into four aliquots. The first aliquot was used as a control (lane 2), the second was treated with Endo H (lane 3), the third was digested with trypsin and chymotrypsin in the absence of Triton X-100 (lane 4), and the last aliquot was digested with trypsin and chymotrypsin in the presence of Triton X-100 (lane 5). After treatment, all samples were subjected to immunoprecipitation with anti-CAT antibody, followed by SDS-PAGE analysis as described under ``Experimental Procedures.'' Note: the results of the fifth aliquot are shown only for the sample X-c. Mb represents microsomes. B, schematic drawings show the orientation of the chimeras inserted into microsomal membranes before and after protease digestion (indicated by +P). Solid hearts and diamonds indicate glycosylated forms and potential N-linked glycosylation sites, respectively. Cleavage sites by signal peptidase are indicated by arrows. sGH and GH represent the signal sequence of rat growth hormone. H, N, and C represent hydrophobic segment, NH termini, and COOH termini, respectively.



The transcripts of plasmids pX-c, pX-1, pX-2, pX-3, and pX-4 were translated in a rabbit reticulocyte lysate in the presence and absence of microsomes. An aliquot of the translation mixture was immunoprecipitated with anti-CAT antibody, and the resulting precipitates were digested with Endo H. Other aliquots were digested with trypsin and chymotrypsin in the presence or absence of Triton X-100, and membrane-protected fragments were recovered by immunoprecipitation with anti-CAT antibody. The results are summarized in Fig. 4A.

The primary translation product of the control chimera X-c gave a single band with a molecular mass of about 33 kDa (X-c, lane 1). The product obtained in the presence of microsomes also gave a single band with a molecular mass of about 33 kDa (X-c, lane 2); however, the molecular mass of this protein was shifted to about 30 kDa by Endo H digestion (X-c, lane 3). After digestion of the translation product with trypsin and chymotrypsin, a 33-kDa protein was recovered by immunoprecipitation (X-c, lane 4), but the translocated protein was digested into undetectable fragments by immunoprecipitation when digested in the presence of Triton X-100 (X-c, lane 5). These results show that, as expected, the cleavable signal sequence of rat growth hormone did very efficiently initiate translocation of the control chimera, and almost all of the translocated proteins were glycosylated and fully protected by membranes from proteolysis.

The primary translation products of the plasmids pX-1, pX-2, pX-3, and pX-4 were about 37, 44, 37, and 47 kDa, respectively (Fig. 4A), and the translation products carried out in the presence of microsomes were about 34, 41, 34, and 44 kDa, respectively. These sizes were not changed by Endo H treatment (Fig. 4A), suggesting that all four chimeric proteins were inserted into membranes where their signal sequences were cleaved and further translocation of their downstream peptides was halted by the hydrophobic segments. Therefore the CAT protein remained in the cytoplasmic side of the membranes. When these translation products were digested with trypsin and chymotrypsin, no fragments were recovered by immunoprecipitation with anti-CAT antibody, which proved that the CAT protein remained in the cytosol and was digested by exogenously added proteases (Fig. 4). Thus, the data have clearly demonstrated that all four hydrophobic segments in the NH-terminal third of subunit have halt transfer signal function when they are in a different context and therefore their nature seems to be intrinsic. In addition, these experiments further confirmed that hydrophobic segments H2 and H4 are transmembrane domains.

DISCUSSION

We report here the topogenesis of the NH-terminal third of the subunit examined by using in vitro ER membrane insertion of two different groups of chimeras. The first group of chimeras encoded fusion proteins consisting of different lengths of truncated subunit with one or more hydrophobic segments located sequentially in the native context and CAT protein as a reporter protein. The second group encoded fusion proteins consisting of NH-terminal 66 amino acid residues (including a cleavable signal sequence) of rat growth hormone, one of the H1, H2, H3, or H4 sequences, and CAT protein. Our data demonstrated that the membrane insertion of the NH-terminal third of subunit was initiated by H1 which functioned as a signal/anchor type II and was achieved by the downstream hydrophobic segments, which acted alternately as halt transfer (H2), insertion (H3), and halt transfer (H4) signals. The data also demonstrated that H1 and H3 were capable of halting translocation of their downstream peptides and that halt transfer function shown by all four hydrophobic segments seemed to be intrinsic.

Homareda et al.(1989) used in vitro ER membrane insertion of truncated subunits that contain two adjacent hydrophobic segments such as H1 and H2 or H3 and H4 to examine which of the hydrophobic segments function as an insertion signal. Since the truncated polypeptides that were inserted into microsomal membranes in an N-ethylmaleimide-sensitive fashion were resistant to alkali extraction, Homareda et al.(1989) have suggested that at least two insertion signals were present in the NH-terminal third of the subunit. The present data confirm the existence of two insertion signals in the NH-terminal third and furthermore demonstrate that H1 and H3 function as a signal/anchor type II.

The number of transmembrane domains in the NH-terminal third of subunit established here is in agreement with those reported previously (Jorgensen et al., 1982; Nicholas, 1984; Farley et al., 1986; Ovchinnikov et al., 1987; Capasso et al., 1992). Orientation of each hydrophobic segment in the lipid bilayer had been predicted as H1 (N and C), H2 (N and C), H3 (N and C), and H4 (N and C) based on the sidedness of the NH terminus (Farley et al., 1986; Ovchinnikov et al., 1987; Ball and Loftice, 1987; Felsenfeld and Sweadner, 1988), the sidedness of the H1-H2 loop (Arystarkhova et al., 1992; Mohraz et al., 1994), the sidedness of the H2-H3 loop (Farley et al., 1986), the sidedness of the H3-H4 loop (Kano et al., 1990), the sidedness of the long COOH-terminal flanking region of H4 (Jorgensen and Collins, 1986; Jorgensen and Andersen, 1988; Farley et al., 1986; Ovchinnikov et al., 1987; see reviews by Vasilets and Schwarz(1993); Lingrel and Kuntzweiler, 1994), and the number of transmembrane domains predicted by hydropathy plot. The present data have confirmed the orientation of H1 and H3 by the occurrence of N-linked glycosylation of the reporter protein at the COOH-terminal flanking region of the hydrophobic segments and sensitivity to protease digestion of the NH-terminal flanking region of the segments.

Although the first group of chimeras showed the orientation of H2 and H4 ( Fig. 2and Fig. 3 ), the results were not conclusive, because the chimeras lacked a marker for the luminal disposition of NH-terminal flanking region of H2 and H4. The conclusion was drawn from the data together with the following circumstantial observations: (a) the COOH-terminal flanking regions of H1 and H3 are located in the ER lumen, (b) the loops between H1 and H2 and particularly between H3 and H4 are too short to span the lipid bilayer, and (c) both loops are highly charged. In order to obtain conclusive results on the orientation, we constructed plasmids encoding chimeras in which one of hydrophobic segments was engineered into a passenger protein that had a cleavable signal sequence with high translocation efficiency. This allowed us to directly examine if these hydrophobic segments function as a halt transfer signal. In this approach, cleavage of an insertion signal was used as a marker for the luminal location of the NH-terminal flanking region of each hydrophobic segment, and digestion of the reporter protein by proteases was used as a marker for the cytoplasmic location of the COOH-terminal flanking region of the segment. As seen in Fig. 4, the control chimera X-c was translocated very efficiently by the cleavable signal sequence of rat growth hormone and almost all of the translocated protein was glycosylated. The apparent molecular mass of the glycosylated protein became smaller by about 3 kDa when digested with Endo H (Fig. 4, X-c: lanes 1 and 3), indicating occurrence of the signal sequence cleavage as expected. Under the same translation/translocation conditions, the signal sequence of the chimeras X-2 and X-4 were cleaved, but the inserted chimeras were not glycosylated. In addition, they were digested by proteases into fragments which did not immunoprecipitate with anti-CAT antibody (see Fig. 4A). These experiments have conclusively confirmed the orientation of H2 and H4. The data have also showed that H1 and H3 are not only a signal/anchor type II but also capable of halting translocation, if employed, in a different context.

The present study has demonstrated that the experimental approaches employed here are very effective in determining the topogenic nature of hydrophobic segments in the native context of a polytopic membrane protein. Using the same approaches, studies on the membrane topology of the COOH-terminal third of Na,K-ATPase subunit are currently in progress.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM20277. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Present address: Dept. of Biochemistry, University of Washington, Seattle, WA 98195.

To whom correspondence should be addressed. Tel.: 212-263-5947; Fax: 212-263-8139.

The abbreviations used are: CAT, chloramphenicol acetyltransferase; DTT, dithiothreitol; Endo H, endoglycosidase H; PAGE, polyacrylamide gel electrophoresis; ER, endoplasmic reticulum.


ACKNOWLEDGEMENTS

We thank Dr. David D. Sabatini for encouragement and support throughout this work. We also thank Dr. Y. Hara for the rat brain Na,K-ATPase -1 subunit cDNA and J. Culkin and F. Forcino for their excellent photographic work. We are very grateful to Dr. Manijeh Mohraz for critical reading of the manuscript and helpful discussion.


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