J. Biochem. 82, 1403-1416 (1977)

Biogenesis of the Mitochondrial Matrix ,

Glutamate Dehydrogenase, in Rat Liver Cells

I. Subcellular Localization, Biosynthesis, and Intracellular Translocation of

Kaname KAWAJIRI, Tomoyuki HARANO, and Tsuneo OMURA

Department of Biology, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka, Fukuoka 812

Received for publication, May 21, 1977

1. The presence of glutamate dehydrogenase in the microsomal fraction of rat liver was confirmed. The identities of mitochondrial and microsomal glutamate dehydrogenases were proved by immunochemical methods and by SDS polyacrylamide gel electrophoresis of purified . 2. Synthesis of glutamate dehydrogenase by the membrane-bound of rough endoplasmic reticulum was determined. Newly synthesized enzyme molecules were discharged on the cytoplasmic surface of endoplasmic reticulum membranes. 3. A precursor-product relationship was found between microsomal and mitochondrial glutamate dehydrogenases. About six hours were needed for the transport of glutamate dehydrogenase from the site of synthesis to mitochondria. 4. The half-life of glutamate dehydrogenase was about 5.5 days, which was somewhat longer than that of mitochondrial total protein determined in the same experiment. 5. Mitochondrial-type was also present in the microsomal fraction. Subfractionation of smooth microsomes revealed the existence of particular light microsomal vesicles in which both glutamate dehydrogenase and malate dehydrogenase were concentrated. These vesicles may participate in intracellular transport of matrix enzymes from microsomes to mitochondria.

Glutamate dehydrogenase (GDH) is functional of amino acids (1). Therefore the biosynthesis in mitochondria as a key enzyme in the and intracellular translocation of this enzyme from the site of synthesis to mitochondria are important not only in the biogenesis of intracellular organelles Abbreviations: GDH, glutamate dehydrogenase; but also in the manifestation of the physiological MDH, malate dehydrogenase; AGG, anti-glutamate function of GDH. dehydrogenase-immunoglobulin; AMG, anti-malate de The interdependence between mitochondrial hydrogenase-immunoglobulin; AOG, anti-ovalbumin immunoglobulin; AAG, anti-serum albumin-immuno protein synthesis and cytoplasmic protein synthesis in the biogenesis of mitochondria is now well globulin; AMPase, adenosine monophosphatase; IgG, immunoglobulin. established (2-7). Synthesis of outer membrane

Vol. 82, No. 5, 1977 1403 1404 K. KAWAJIRI, T. HARANO, and T. OMURA

proteins (8) and some protein components of the genizer to give a 15% homogenate. The homog inner membrane (9) by cytoplasmic ribosomes enate was centrifuged at 600 •~ g for 10 min to has been repeatedly shown. Mitochondria] matrix remove nuclei and cell debris. The supernatant proteins, which constitute about 50% of the total was centrifuged at 5,000 •~ g for 15 min to pre mitochondrial proteins, are also synthesized on cipitate mitochondria. The post-mitochondrial cytoplasmic ribosomes (10-12). In the case of supernatant was centrifuged at 10,000 •~ g for matrix enzymes, they must be transported from 15 min to remove lysosomes. The post-lysosomal the cytoplasm into the mitochondrial inner com supernatant was centrifuged at 170,000 •~ g for partment across two membranes, the outer and 60 min to precipitate microsomes. Both the inner membranes. mitochondria and the microsomes were washed The purpose of this study was to elucidate once with 0.25 M sucrose-10 mm Tris-HCl-1 mm the mechanism of intracellular transfer of GDH EDTA (pH 7.5). To prepare rough and smooth from its site of synthesis to the mitochondria) microsomes, 15 ml of the post-lysosomal super inner compartment. We confirmed the presence natant were layered over 15 ml of 1.3 M sucrose of GDH in the microsomal fraction obtained from 10 mm Tris-HCl-1 mm EDTA (pH 7.5). After rat liver. We also demonstrated the synthesis of 4 h centrifugation at 170,000 •~ g, the layer of GDH by the membrane-bound ribosomes of rough smooth microsomes which formed between the endoplasmic reticulum. The GDH in the micro 0.25 M sucrose and the 1.3 M sucrose was carefully somal fraction was present on the outside surface separated from the tightly packed pellet of rough of microsomal vesicles, which coincided with the microsomes, and diluted five to six fold with observation that the nascent peptides of the enzyme 0.25 M sucrose-10 mm Tris-HCl-1 mm EDTA (pH were discharged from the ribosomes to the cyto 7.5). The pellets were also suspended in the plasmic surface of endoplasmic reticulum. As a same sucrose solution and the suspensions were precursor-product relationship was found between centrifuged at 170,000 •~ g for 60 min to sediment microsomal and mitochondrial GDHs, the enzyme the rough and smooth microsomes. Both micro associated with microsomal particles is a pool of somal fractions were washed once with the same newly synthesized molecules to be transported into sucrose solution. mitochondria. Subfractionation of Smooth Microsomes by Butow and Kellems reported the existence of Sucrose Density Gradient Centrifugation-The cytoplasmic polyribosomes in association with the smooth microsomes (about 16 mg protein) were cytoplasmic face of the outer mitochondria) layered over a continuous concentration gradient membrane in yeast cells, and they suggested the of sucrose from 0.7 M to 1.25 M containing 10 mm vectorial discharge of nascent peptides from the Tris-HCl (pH 7.5). Centrifugation was performed ribosomes, which ensures their transfer into the with a Hitachi SW 25 rotor at 25,000 rpm for mitochondrial inner compartment (13-16). Our 3 h. One ml fractions were collected from the results indicate, however, the principal role of bottom of tube. membrane-bound ribosomes of rough endoplasmic Purification of GDH from Rat Liver Mito reticulum in the biosynthesis of mitochondria) chondria. -GDH was purified from rat liver mito matrix proteins. chondria by the following procedure. The enzyme was solubilized by sonic oscillation of washed

mitochondria at 4•Ž for 60 s. The sonicated MATERIALS AND METHODS suspension was centrifuged at 105,000 •~ g for Preparation Mitochondria, Microsomes, Rough, 60 min. The supernatant containing about 3,000 and Smooth Microsomes from Rat Liver-Sprague mg of protein was concentrated by freeze-drying Dawley male rats weighing 200g to 300g were and applied to a column (3.7 •~ 80 cm) of Sepharose used. They were fasted overnight and then 4B equilibrated with 10 mm phosphate-1 mm killed. Their livers were excised, perfused thor EDTA-20 ƒÊM ADP (pH 7.5). ADP was added oughly with ice-cold 0.9 % NaCl, and homogenized in this step to stabilize the enzyme. The column in 0.25 M sucrose-10 mm Tris-HCl-1 mm EDTA was eluted with the same buffer, and the eluate (pH 7.5) with the aid of a Potter glass-Teflon homo- was collected in 15 ml fractions. The GDH

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (1) 1405

rich fractions were combined, and applied to a

column (2.7 •~ 20 cm) of DEAE-Sephadex A-50

equilibrated with 10 mm phosphate-1 mm EDTA

(pH 7.0). The column was washed with the same buffer, and then eluted with an increasing linear

concentration gradient of KCl from 0 to 0.4 M

in the same buffer system. The eluate was col

lected in 10 ml fractions. The GDH-rich fractions

were combined and dialyzed against 10 mm

phosphate-1 mm EDTA (pH 7.0) overnight. The dialyzed solution was then applied to a column

(1.4 •~ 15 cm) of hydroxylapatite equilibrated with 10 mm phosphate (pH 7.0). After washing with

the same buffer, the enzyme was eluted with an

increasing linear concentration gradient of phos Fig. 1. SDS polyacrylamide gel electrophoresis of phate from 10 mm to 0.4 M (pH 7.0). The eluted GDH was concentrated with a collodion bag. purified mitochondrial and microsomal glutamate de hydrogenases. (A) Purified GDH from rat liver mito About 20 mg of purified enzyme was recovered chondria was analyzed by electrophoresis with 5% and its activity was about 170 units per mg of polyacrylamide gel of pH 8.5. Three ƒÊg of enzyme protein. The purification ratio and the recovery protein were applied on the column. (B) A mixture of were about 150 and 34% from whole mitochondria, mitochondrial and microsomal GDH was analyzed as respectively. The purified GDH gave a single described in (A). A mixture of 2 ƒÊg of mitochondrial

band when examined by SDS polyacrylamide gel enzyme and 1ƒÊg of microsomal enzyme was applied

electrophoresis (Fig. 1). on the column. (C) Purified GDH from rat liver micro Purification of GDH from Microsomes somes was analyzed in the same way. One ƒÊg of enzyme was applied on the column. Purification of GDH from rat liver microsomes was carried out as follows. Microsomes were

suspended in 0.15 M KCl to release the microsomal immunoglobulin fraction was prepared by frac

GDH. The purification procedure of GDH tionation of the serum with ammonium sulfate was almost the same as that for the mitochondrial (25-45 %) and then dialyzed against 50 mm phos enzyme except DEAE-Sephadex column chro phate buffer (pH 7.5). The quantitative precipita

matography was replaced by chromatography on tion reaction was carried out as described in the a phospho-cellulose column, which was equilibrated legend of Fig. 3. Antisera against ovalbumin and

with 10 mm phosphate-1 mm EDTA (pH 7.0) and mitochondrial malate dehydrogenase were pre eluted with a concentration gradient of KCl from pared in the same way. 0 to 0.4 M in the same buffer system. Purified Preparation of Antibody-Sepharose Gel-The

GDH from microsomes gave a single band when conjugation of immunoglobulin with Sepharose examined by SDS polyacrylamide gel electro 4B was carried out according to Porath et al. (17)

phoresis (Fig. 1). with some modifications. Six grams of cyanogen Preparation of Antibody against GDH-Male bromide were added to 60 g (wet weight) of

white rabbits, which were generously supplied by Sepharose 4B. The pH was kept between 11 and

Takeda Chemical Industries, Ltd. Osaka, were 12, and the mixture was stirred for 10 min in

immunized with purified mitochondrial GDH 0.1 M NaHCO3. After washing with distilled

in the following way. The animals were injected water and 0.1 M NaHCO3, the activated Sepharose

with 3 mg of purified GDH mixed with I ml of 4B gel was divided into three parts and each part

Freund's complete adjuvant (Difco, Co). Three was suspended in 25 ml of 0.1 M NaHCO3. About

weeks later, 1 mg of purified GDH was injected 100 mg of anti-GDH-immunoglobulin (AGG), intravenously. Blood was collected from the anti-malate dehydrogenase-immunoglobulin

immunized animals one week after the last injection, (AMG), and anti-ovalbumin-immunoglobulin and serum was obtained from the blood. The (AOG) were added to each portion of the gel.

Vol. 82, No. 5, 1977 1406 K. KAWAJIRI, T. HARANO, and T. OMURA

The mixtures were left at 4•Ž overnight, and the combined, and layered over a discontinuous con

gels were then washed with 0.1 M NaHCO3 and centration gradient of sucrose from 1.3 M to 2.1 M

suspended in the same solution containing 0.05 M containing medium A. After centrifugation at ethanolamine to block excess activated groups. 170,000 •~ g for 3.5 h in a Hitachi RP 50 rotor,

The Sepharose 4B gels thus conjugated with AGG, smooth microsomes, rough microsomes, and free AMG, and AOG will be called AGG-Sepharose, ribosomes were clearly separated from each other.

AMG-Sepharose, and AOG-Sepharose, respec The pellets of free ribosomes were suspended tively. The three antibody-Sepharose gels were in 15 ml of medium A. The rough microsomal

suspended in 20 mm phosphate buffer (pH 7.5) fraction was diluted with 30 ml of medium A.

and used in the following immunoadsorption Equal volumes of 8 % Triton X-100-2% DOC

experiments. 0.6 M KCl were added to the suspensions of free

Immunoadsorption of Smooth Microsomes to ribosomes and rough microsomes. The mixtures

Antibody-Sepharose Gel-AMG-(AGG-, AOG-) were layered over 2.1 M sucrose containing medium Sepharose was incubated with smooth microsomes A and were centrifuged at 170,000 •~ g for 2 h.

at 0•Ž for 1 h. Then the reaction mixture was The pellets of free ribosomes and membrane

centrifuged at 3,000 rpm for 10 min, and the bound ribosomes were washed separately with supernatants were removed by Pasteur pipette medium A and suspended in 50 mm Tris-HC1

to measure the amounts of un-adsorbed microsomes (pH 7.6)-15 mM EDTA-0.9 % NaCl. After stand and microsomal enzyme activities. The amounts ing for 30 min, the mixture was centrifuged at

of antibody-Sepharose and microsomes are given 170,000 •~ g for 2 h. Ten ml of supernatant

in the legends of figures and tables. were taken out and Triton X-100 was added to a

Determination of Site of Synthesis of GDH final concentration of 0.2%. RNase inhibitor

by Immunoprecipitation of Radioactive Nascent (20) was included in the media used in the above Peptides-(i) Peptic digestion of AGG and AOG: steps. AOG-F(ab)2 (10 mg) and ovalbumin (0.2

AGG and AOG were digested with pepsin to obtain mg) were added to the mixture and it was incubated

the F(ab)2 as described by Nisonoff (18). One to at 4•Ž overnight. The reaction mixture was

two percent solutions of AGG and AOG were centrifuged at 3,000 rpm for 10 min, and the

incubated with pepsin at a protein to enzyme supernatant divided into two parts. AOG-F(ab)2 ratio of between 100: 1 and 100 : 2 in 50 mm was added to one portion and AGG-F(ab)2 to

acetate buffer (pH 4.5) at 37•Ž for 18 h. Frac the other portion. After 30 min incubation at

tionation of the peptic digests of AGG and AOG 25•Ž, ovalbumin (0.1 mg) or GDH (60ƒÊg) was

was carried out by gel filtration with a Sephadex added to each portion as the carrier. After 1 h

G-100 column according to Ustumi and Karush incubation at 37•Ž, the reaction mixture was left

(19). at 4•Ž overnight. Then the mixtures were cen

(ii) Immunoprecipitation of 3H-labelled nascent trifuged at 3,000 rpm for 10 min, and the pellets peptides with F(ab)2: Five male rats, weighing were washed four times with 0.9% NaCl-10 mM 160 g to 180 g were starved overnight. 3H-Leucine KP1 (pH 7.6), and dissolved in 1 N NaOH. The

was injected intravenously into the rats through radioactivity was counted by a Packard liquid

caudal veins in amounts of 100pCi/100 g of body scintillation counter using toluene-Triton X-100 weight of the rats. Two minutes after the injec scintillant.

tions, the rats were killed and the livers removed Determination of Site of Synthesis of GDH

quickly. The livers were homogenized in 3 vol. by the Binding of 1251-Labelled Fab Monomer with of 0.88 M sucrose containing medium A (50 mm Polysomes-(i) Preparation of 1231-labelled Fab:

Tris-HCl (pH 7.6)-10 mm magnesium acetate To obtain the Fab monomer, AGG and AOG 25 mm KCl). The homogenate was centrifuged were digested with papain according to the method

at 10,000 x g for 20 min to remove nuclei, mito of Porter (21). Iodination of AGG and AOG

chondria, and lysosomes. The resulting pellets Fab monomers was carried out according to the

were resuspended in the same sucrose solution method of Sonoda and Schlamowitz (22). 125- and centrifuged at 10,000 x g for 20 min. The labelled Fab monomers of AGG and AOG thus first and second post-lysosomal supernatants were prepared had specific activities of 2.05 •~ 105 cpm/mg

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (1) 1407

and 1.44 •~ 105 cpm/mg, respectively. above. GDH was released from both microsomal

(ii) Binding of 7251-labelled Fab monomer fractions by washing them with 10 mM Tris-HC with polysomes: Free and membrane-bound poly l 0.3 M KCl (pH 7.5). Mitochondrial GDH was

somes were isolated as described above. Polysomes solubilized by sonication. Mitochondrial and

were incubated with 57ƒÊg of AGG125I Fab mono microsomal GDHs were purified as described

mer or with a similar amount of AOG-125I Fab. above. The purified enzymes were precipitated

The amounts of polysomes are given in Fig. 6 in by adding 10% TCA and the precipitates were terms of mg of protein. The incubation was washed with a mixture of ethanol-ether (3 : 1).

carried out at 0•Ž for 30 min in 20 mm Tris-HCl The washed precipitate was dissolved in 1 N NaOH.

0.9 % NaCl (pH 7.6). The mixture of polysomes The radioactivity was counted with a Packard

and antibody preparations were centrifuged at liquid scintillation counter using toluene-Triton

170,000 •~ g for 1 h to remove unreacted 725I-Fab X-100 scintillant.

monomer. The precipitated polysomes were Turnover Experiment-Male rats weighing

washed once with the same buffer, and the radio 190 g to 210 g were used. DL-14C-Leucine was

activity of the washed polysomes was counted by injected intraperitoneally into the rats in amounts an auto-gamma scintillation spectrometer. of 10 ƒÊCi/100 g of body weight of the animals.

Direction of Discharge of GDH Nascent Each time-point represents the pooled livers of

Peptides Synthesized by Membrane-Bound Ribo four animals. Mitochondria were isolated as described above and GDH was solubilized and somes-The direction of discharge of nascent

peptides of GDH was investigated by the following purified. Analytical Procedures-The cytochrome b5 two separate methods. and cytochrome P-450 contents in microsomes (i ): Rough microsomes (15 mg of protein) were determined as described by Omura and were incubated with low concentrations of trypsin Sato (23). NADPH-cytochrome c reductase ac (0.01% and 0.1%) at 0•Ž for 20 min. After tivity was assayed as described previously (24). incubation, excess amounts of trypsin inhibitor NADH-cytochrome b5 reductase was assayed by (final concentration 0.2%) and an equal volume of measuring its NADH-ferricyanide reductase ac 8%. Triton X-100-2% DOC-0.6 M KCl were tivity (25). Arylacylamidase was assayed as added. Ribosomes were isolated as described above. Binding of 125I-labelled Fab monomer described by Akao and Omura (26). Sulfite

with ribosomes isolated from trypsin-treated oxidase activity was assayed as described by

rough microsomes or from non-treated rough Cohen and Fridovich (27) using cytochrome c as the electron acceptor. GDH was measured microsomes was carried out as described above. according to Arnold (28) with some modifications (ii): Nascent peptides of rough microsomes in 0.1 M Tris-HCI-1 mm EDTA (pH 8.2) containing were labelled in vivo by injecting 3H-leucine into 0.1 mm NADH, 50 mm CH3000NH4, 100 ƒÊM the rats and isolated rough microsomes were ADP, 3 mm ƒ¿-ketoglutarate. Malate dehydro incubated with trypsin at 0•Ž for 20 min. After

incubation, ribosomes were isolated as described genase (MDH) was measured in 0.1 M KPi (pH 7.5) containing 0.1 mm NADH, 200ƒÊM oxaloace above. Radioactive nascent peptides were re tate. The assay system for AMPase contained leased with EDTA from the ribosomes, and 5 mm AMP, 5 mm MgCl2, 0.1 M Tris-HCl (pH 7.5) immunoprecipitation with F(ab)2 was carried out and microsomes in a final volume of 0.5 ml. After as described above. incubation for 20 min at 30•Ž, 0.5 ml of 10 Incorporation of Radioactive Leucine into TCA was added to stop the reaction. Inorganic Microsomal and Mitochondrial GDHs-Male rats

weighing 190 g to 210 g were used. L-14C-Leucine phosphate liberated was determined by the method of Fiske and SubbaRow (29). UDP-galactose was injected intravenously into the rats through transferase was assayed as described by Beaufay caudal veins in amounts of 10 fiCi/100 g of body et al. (30). Protein was determined by the method weight of the rats. Each time-point represents of Lowry et al. (31) using bovine serum albumin the pooled livers of five animals. Mitochondria, as the standard. RNA was determined using rough microsomes, and smooth microsomes were Mejbaum's orcinol method (32). Phospholipid prepared from the pooled livers as described

Vol. 82, No. 5, 1977 1408 K. KAWAJIRI, T. HARANO, and T. OMURA phosphorus was determined by Allen's procedure activation by sonication or detergent treatment, (33) and the value obtained was multiplied by whereas the GDH activity of microsomes was 25 to obtain the amount of phospholipid. Disc detectable without such treatment and activated polyacrylamide gel electrophoresis in the presence only by about 30% by the addition of Triton of sodium dodecyl sulfate was carried out by X-100. GDH activity was not detectable in the the method of Weber and Osborn (34). The . following proteins with known molecular weights Identity of Mitochondrial and Microsomal were used in determining the molecular weight of GDHs-The properties of mitochondrial and GDH; pepsin (34,000), ovalbumin (44,000), IgG microsomal GDHs were compared by an immuno H-chain (52,000), catalase (57,500), and bovine chemical method and by SDS polyacrylamide gel serum albumin (68,000). electrophoresis. Purified mitochondrial and Reagents and Biochemicals-L-(4,5-3H)-leucine, microsomal enzymes gave a single band when L-(1-14C)-leucine, DL-( 1-14C)-leucine, and 1251-KI examined by SDS polyacrylamide gel electro were purchased from Daiichi Pure Chemicals phoresis (Fig. 1). Furthermore, a mixture of Co., Ltd., Tokyo. Crystalline yeast cytochrome mitochondrial and microsomal enzymes also c was kindly supplied by Sankyo Co., Ltd., Tokyo. gave a single band (Fig. 1). The molecular weight Other chemicals were of reagent grade. of the subunit of the dehydrogenase was determined to be about 62,000 from the mobility on the gel

RESULTS (Fig. 2). Figure 3 shows the quantitative pre cipitation reactions of mitochondria] and micro Intracellular Localization of GDH in Rat somal GDHs against AGG. The quantities of Liver Cells-Table I shows the distribution of precipitated protein and enzyme activity recovered GDH in the mitochondrial and microsomal in the supernatant in the regions of antigen excess fractions. Succinate-cytochrome c reductase and were identical for the mitochondrial and micro NADPH-cytochrome c reductase were used as somal enzymes. The results of Fig. 1 and Fig. 3 marker enzymes for mitochondria and microsomes, indicate the identity of microsomal and mito respectively. The relative specific activity (RSA) chondrial GDHs. value of GDH in microsomes was significantly Intrainicrosomal Localization of GDH-Figure higher than that of succinate-cytochrome c reduc 4 shows the effect of KCl on the release of micro tase in the same fraction. GDH activity in micro somal enzymes from the membranes. As shown somes could not be explained by the contamination in this figure, GDH was released from microsomes of the microsomal fraction by mitochondria. with low concentrations of KCl. Figure 5 shows The mitochondrial GDH was inactive without the effect of AGG and AOG on the activity of

TABLE I. Distribution of glutamate dehydrogenase in rat liver mitochondria and microsomes. The distribu tions of enzymes between mitochondrial (Mt) and micro somal (Ms) fractions are expressed as their relative specific activities (RSA). 100% of RSA's of glutamate dehydrogenase, NADPH-cytochrome c reductase, and succinate-cytochrome c reductase were 1.08 U/mg, 0.18 U/mg, and 0.16 U/mg, respectively.

Fig. 2. Determination of molecular weight of glutamate dehydrogenase by SDS polyacrylamide gel electrophore sis. The molecular weight of GDH was determined by SDS polyacrylamide gel electrophoresis. Marker pro teins used were: a, pepsin (34,000); b, ovalbumin (44,000); c, IgG H-chain (52,000); d, catalase (57,500); e, bovine serum albumin (68,000).

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (I) 1409

Fig. 3. Quantitative immunoprecipitation of glutamate dehydrogenase by AGG. The antigen used was partially purified enzyme at the Sepharose 4B step. To 0.1 ml of AGG was added varying amounts of mitochondrial and microsomal enzymes as indicated in the figure. The reaction mixture, in a final volume of 1.5 ml of 0.9% NaCl-20 mm KPi (pH 7.5), was incubated at 37•Ž for 60 min and then stored at 4•Ž for 48 h. After centrifugation of the reaction mixture, the supernatant was assayed for GDH as described in " MATERIALS AND METHODS." The precipitate was washed three times with 0.9% NaCI-20 mm KPi (pH 7.5) and assayed for protein. Circles denote protein in immuno

precipitates and triangles denote enzyme activity in supernatant. •ü, ƒ¢, GDH partially purified from microsomes; •œ, •£, GDH partially purified from mito chondria.

microsomal GDH. The microsome-bound GDH TABLE II. Precipitation of 3H-leucine-labelled nas was inhibited by AGG to the same extent as when cent peptides with AGG. Pulse labelled nascent peptides partially purified mitochondrial GDH was used. were released from membrane-bound and free ribosomes This reactivity between antibody and membrane (Rs) with EDTA, and immunoprecipitation was carried out with pepsin-digested antibodies as described in bound antigen in intact microsomes suggests the " MATERIALS AND METHODS ." To determine the outside surface location of GDH in the isolated location of GDH nascent peptides in microsomal vesi microsomal vesicles. cles, rough microsomes (Ms) were incubated with 0.1 Site of Synthesis of GDH in Liver Cells of trypsin at 0•Ž for 20 min. The site of synthesis of GDH in liver cells was investigated by the following two methods. (i) Immunoprecipitation of 3H-leucine-labelled nascent peptides of GDH by AGG: 3H-Leucine labelled nascent peptides were released with EDTA from free and membrane-bound ribosomes. Table II shows the precipitation of nascent GDH peptides by AGG. In order to minimize non specific precipitation of radioactivity, a pepsin digested fragment (F(ab)2) of AGG was used. The radioactivity of nascent peptides precipitated

Vol. 82, No. 5, 1977 1410 K. KAWAJIRI, T. HARANO, and T. OMURA

by AGG was significantly higher with membrane

bound ribosomes.

(ii) Binding of 1251-labelled Fab monomer of AGG with free and membrane-bound ribosomes:

Free and membrane-bound ribosomes were incu

bated with 125I-labelled Fab monomer of AGG,

and the ribosomes were sedimented by centrifuga

tion at 170,000 •~ g for 2 h. As shown in Fig. 6,

larger amounts of labelled Fab monomer were

bound with membrane-bound ribosomes than with free ribosomes when a fixed amount of 125I-Fab was incubated with varying amounts of

Fig. 4. Release of microsomal enzymes from mem brane with KCl. A fixed amount of microsomes (7 mg) was incubated with varying concentrations of KCl in final volumes of 5 ml for 30 min at 4•Ž. The reaction mixture was then centrifuged at 105,000 •~ g for 60 min, and enzyme activities in the supernatant were assayed. The 100% level indicates the enzyme activity in the whole reaction mixture. •ü, MDH; •œ, GDH; •~, NADPH cytochrome c reductase and sulfite-cytochrome c reductase.

Fig. 5. Effect of AGG and AOG on the activity of

glutamate dehydrogenase of rat liver microsomes. Microsomes were preincubated with the indicated Fig. 6. Binding of 125I-labelled Fab monomer of AGG amounts of AGG for 10 min at 25•Ž in 0.1 M Tris with free (A) and membrane-bound (B) ribosomes. HCI-1 mm EDTA (pH 8.2). AOG was used as a con Fixed amounts of 1251-Fab monomer of AGG and AOG trol immunoglobulin preparation which has no specific were incubated with varying amounts of free and interaction with microsomes. After incubation, GDH membrane-bound ribosomes, which are shown in the activity was assayed. The effect of AGG on partially figure as mg of protein, at 0•Ž for 30 min in 20 mM Tris-HCl-0.9% NaCl (pH 7.6). After incubation, ribo purified mitochondrial GDH is also shown in the figure. The GDH activities of microsomes and the partially somes were pelletted by centrifugation at 170,000 •~ g

purified preparation were the same (0.05 U) in the for 2 h. Radioactivities of the ribosomal pellets were absence of serum. •œ, Microsomes and AGG; -•ü-, measured by an auto-gamma scintillation spectrometer. •œ

partially purified mitochondrial GDH 'and AGG, , AGG; •ü, AOG. The binding of AOG with ribo --•ü--, microsomes and AOG. somes indicates non-specific attachment of Fab.

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (I) 1411 ribosomes. Table II and Fig. 6 indicate that GDH is synthesized by membrane-bound ribosomes and not by free ribosomes in rat liver cells. Direction of Discharge of GDH Nascent Peptides from Membrane-Bound Ribosomes-The following experiment was designed to elucidate whether newly synthesized peptides of GDH were discharged on the outside surface of the endoplasmic reticulum membrane or discharged in the lumen. Rough microsomes were first treated with trypsin. Then ribosomes were isolated and incubated with 1251-labelled Fab monomer of AGG. If newly synthesized molecules of GDH were discharged on the outside surface of endo plasmic reticulum, its nascent peptides should be susceptible to digestion with trypsin, and the binding of 125I-labelled antibody to ribosomes would decrease. Figure 7 shows that nascent peptides of GDH were digested with trypsin, whereas nascent peptides of serum albumin, which are known to be discharged in the lumen of endo plasmic reticulum, were not affected by trypsin digestion. It is concluded that newly synthesized molecules of GDH are discharged on the cyto plasmic surface of the endoplasmic reticulum. This conclusion is in agreement with that ob tained from the immunoprecipitation experiments (Table II).

Fig. 8. Subfractionation of smooth microsomes by sucrose density gradient centrifugation. Subfractiona tion of smooth microsomes by sucrose density gradient centrifugation was carried out as described in " MA TERIALS AND METHODS." The direction of sedi Fig. 7. Binding of 125I-labelled Fab monomer of AGG mentation is from right to left. The concentration with membrane-bound ribosomes isolated form trypsin gradient of sucrose was from 0.7 M to 1.25 M. The treated rough microsomes. Trypsin digestion of rough enzyme activities of subtractions are expressed as their micr somes was carried out at 0•Ž for 20 min. Bound percentages of the highest values. (A) Distribution of ribosomes were isolated from trypsin-treated rough NADPH-cytochrome c reductase (--•œ --), and GDH microsomes as described in "MATERIALS AND (•ü) among subfractions. (B) Distribution of MDH METHODS." Binding of 125I-labelled antibody mon (•ü) and total microsomal protein (--•œ--) among sub fractions. (C) Distribution of AMPase (-•œ-) and omer was carried out as described in the legend of Fig. 6. UDP-galactose transferase (•ü) among subfractions. •œ,AGG; •ü, AAG.

Vol. 82, No. 5, 1977 1412 K. KAWAJIRI, T. HARANO, and T. OMURA

Subfractionation of Smooth Microsomes by reductase (Fr. I) and in the other GDH (Fr. II) Sucrose Density Gradient Centrifugation-In order showed high activity. Chemical compositions to determine whether GDH molecules located on and typical microsomal enzyme contents of these the outside of microsomal vesicles are distributed two fractions are identical. As the GDH-rich uniformly among the vesicles or not, we carried fraction contained cytochrome b5 and cytochrome out the subfractionation of smooth microsomes P-450, it is likely that it was also derived from by sucrose density gradient centrifugation. Figure endoplasmic reticulum. 8 (A) shows the distributions of NADPH-cyto Separation of GDH and MDH-Rich Smooth chrome c reductase and GDH among the subfrac Microsomal Vesicles by Immunoadsorption Method tions. Figure 8 (B) shows the distributions of -Glutamate and malate dehydrogenases are MDH and total microsomal protein. As is shown located on the outside surfaces of microsomal in Figs. 8 (A) and (B), the peak of GDH as well vesicles and concentrated in particular smooth as that of MDH was present in lower sucrose microsomal vesicles. If the presence of these density portions than NADPH-cytochrome c reductase, which is a typical marker enzyme of microsomes. Figure 8 (C) shows the distributions of AMPase and UDP-galactose transferase, which are the marker enzymes of plasma membranes and Golgi apparatuses, respectively. The peaks of these two enzymes were in the same position as that of NADPH-cytochrome c reductase. It is therefore unlikely that GDH-rich vesicles in lower sucrose density portions originated by the fragmentation of plasma membranes or Golgi apparatuses. Table III shows the chemical and enzymic compositions of two microsomal sub fractions in one of which NADPH-cytochrome c

TABLE III. Comparison of enzymic and chemical compositions among microsomal subfractions. Sub fractionation of smooth microsomes (s-Ms) was carried out as described in the legend of Fig. 8. Fr. I and Fr. II represent the subfractions in which NADPH-cyto chrome c reductase and glutamate dehydrogenase show ed highest specific activities, respectively.

Fig. 9. Immunoadsorption of mitochondrial matrix

proteins to antibody-Sepharose. Varying amounts of mitochondrial sonic extract (10 mg/ml) were incubated with 200 mg of AMG-Sepharose, AGG-Sepharose, or AOG-Sepharose in 2 ml of 20 mm potassium phosphate buffer (pH 7.5). The incubation conditions are de scribed in " MATERIALS AND METHODS." After incubation, the mixture was centrifuged, and the ac tivities of GDH (A) and MDH (B) in the supernatant were assayed. •~, AOG-Sepharose; •ü, AMG-Sepha rose; •œ, AGG-Sepharose.

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (1) 1413 enzymes on microsomal vesicles represents a cytochrome c reductase were adsorbed to the gel. transient state of their intracellular migration These results indicated that MDH and GDH from the site of synthesis to mitochondria , informa were present on the same microsomal vesicles. tion about their distribution on the surface of The results of another experiment in which treat endoplasmic reticulum is important. We attempted ment with AMG-Sepharose was repeated are the separation of microsomal vesicles by immuno shown in Table IV. The adsorption of MDH adsorption method (35) to see whether GDH and GDH activities was about 50% in the first and MDH are associated with the same vesicles adsorption treatment. The un-adsorbed smooth or not. We used Sepharose 4B gels conjugated microsomes were incubated again with fresh AMG with AMG and AGG. A sonic extract of mito Sepharose, and the enzyme activities in the super chondria was incubated with AMG-Sepharose, natant assayed. The activities of MDH and GDH and we observed complete adsorption of MDH were not decreased by the second treatment. As while GDH was not adsorbed at all (Fig. 9B). not more than 50% of the MDH and GDH could On the other hand, the adsorption of GDH to be removed by immunoadsorption of microsomes AGG-Sepharose was much less efficient although to AMG-Sepharose, it is likely that the adsorption the specificity of AGG-Sepharose for GDH was represented the selective removal of particular evident (Fig. 9A). Figure 10 shows the results of microsomal vesicles which contained larger amounts a typical experiment in which a fixed amount of of MDH and GDH on the outside. In fact, AMG-Sepharose was incubated with varying when smooth microsomes were incubated with amounts of smooth microsomes. The amounts AMG-Sepharose and un-adsorbed microsomes of un-adsorbed microsomal enzyme activities were subfractionated by sucrose density gradient were determined. Although AMG-Sepharose was centrifugation, the GDH rich fraction disappeared. used in the experiment shown in Fig. 10, both Intracellular Transport of GDH in Liver Cells MDH and GDH activities were adsorbed to the In order to determine whether GDH in microsomes same extent, about 50%, whereas only 10-15 is a precursor pool participating in the transport of NADPH-cytochrome c reductase and sulfite of this enzyme to mitochondria, we determined the incorporation of 14C-leucine in vivo into mito chondrial and microsomal GDHs, and the results are shown in Fig. 11. GDH purified from rough microsomes acquired a very high specific radio activity 10 min after the injection, and the radio activity decreased gradually. Conversely, GDH

TABLE IV. Repeated treatment of smooth micro somes with AMG-Sepharose. Eight mg of smooth microsomes were incubated with 200 mg of AMG Sepharose. The incubation conditions are described in the text. After incubation, the mixture was centrifuged, and the supernatant was again incubated with the same amount of fresh AMG-Sepharose. Fig. 10. Immunoadsorption of smooth microsomes to AMG-Sepharose. Varying amounts of smooth micro somes were incubated with 200 mg of AMG-Sepharose in 2 ml of potassium phosphate buffer (pH 7.5). The incubation conditions are described in " MATERIALS AND METHODS." After incubation, the mixture was centrifuged, and the enzyme activities in the super natant were assayed. The amounts of microsomes are given in the figure in terms of mg of protein. (-•œ-) GDH, (-•ü-) MDH, (--•œ--) sulfite-cytochrome c reductase, (--•ü --) NADPH-cytochrome c reductase.

Vol. 82, No. 5, 1977 1414 K. KAWAJIRI, T. HARANO, and T. OMURA

purified from mitochondria showed a low specific radioactivity at 10 min, and the radioactivity increased up to 6 h. Based on this result, it is concluded that GDH in microsomes is the precursor of the mitochondrial enzyme. Turnover of Mitochondrial GDH in Liver Cells-Figure 12 shows the results of a turnover experiment in which mitochondrial total protein, sonic solubilized protein, and purified GDH were examined. After the in vivo labelling by intra peritonea] injection of radioactive leucine into the rats, a mitochondrial fraction was prepared from the liver at each-time point, and GDH was ex tracted and purified. The half-lives of GDH and mitochondrial total protein were graphically estimated from the figure to be about 5.5 days and 4.5 days, respectively.

Fig. 11. Specific radioactivity of glutamate dehydrogen DISCUSSION ases purified from rough and smooth microsomes, and mitochondria after a single injection of 14C-leucine. The Mitochondria are formed by close cooperation radioactive amino acid was injected into the rats at time between the cytoplasmic and mitochondria) protein 0, and five animals were killed at each time-point to synthesizing systems (2-7). It has been confirmed prepare rough and smooth microsomal fractions and in recent years that some subunits of several mitochondria from their pooled livers as described in the mitochondria) inner membrane enzymes are made text. GDH was purified from these fractions and radio by the protein synthesizing system in mitochondria activities of the purified enzymes were counted. GDH (36-38), but the contribution of the system in the purified from rough microsomes (-•œ-), smooth micro synthesis of total mitochondrial protein is estimated somes (--•œ--), and mitochondria (•ü). The radio to be only about 10% (39, 40). Therefore the activity is shown in the figure in terms of dpm per mg of majority of mitochondrial proteins are made in protein. the extramitochondrial space by cytoplasmic ribosomes, and subsequently localized to mito chondria by some regulatory mechanism. Studies on mitochondrial biogenesis have so far been mostly concerned with the formation of the inner mem brane, but selective localization of matrix enzymes and enzymes into those particular mitochondrial compartments is also an important problem to be considered. The major sites of protein synthesis in the cytoplasmic space are membrane-bound ribosomes of rough endoplasmic reticulum and free ribosomes. Fig. 12. Decay of specific radioactivity of mitochon It is generally accepted that membrane proteins drial total protein, sonic extract, and glutamate de and secretory proteins are exclusively made by hydrogenase. DL-14C-Leucine was injected intraperito membrane-bound ribosomes whereas most cytosol neally into rats in an amount of 10ƒÊCi/100 g of body weight. Each time-point represents the pooled livers proteins are made by free ribosomes (41). In this of four animals. Arrows denote half-lives determined study, the site of synthesis of GDH was determined graphically. The radioactivity is shown in the figure by two separate methods, and it was concluded in terms of dpm per mg of protein. •ü; Mitochondrial that GDH is synthesized on membrane-bound total protein; •£, sonic extract; •œ, GDH. ribosomes. It was also confirmed that nascent

J. Biochem. BIOGENESIS OF GLUTAMATE DEHYDROGENASE IN RAT LIVER CELLS (I) 1415

peptides of GDH are discharged on the cytoplasmic In order to investigate if microsomal GDH surface of endoplasmic reticulum. These observa is uniformly distributed over the endoplasmic tions agree with the outside location of GDH in reticulum, subfractionation of microsomes was microsomal vesicles. carried out. Smooth microsomes were used in Butow et al. (13-16) have recently shown the experiment since the vesicles of rough micro that some cytoplasmic ribosomes in yeast cells somes are heterogeneous owing to variations in exist in association with the cytoplasmic face of the amounts of attached ribosomes (51) and it the outer mitochondrial membrane, and they was not possible to detach ribosomes from micro suggested the possibility that some mitochondrial somal membranes without releasing GDH. Kuwa proteins are synthesized by particular cytoplasmic hara and Ito reported that sulfite-cytochrome c ribosomes on the mitochondria) outer membrane reductase and were distributed in and the newly synthesized proteins are discharged higher sucrose density portions than NADPH across mitochondrial membranes as in the vectorial cytochrome c reductase when smooth microsomes translocation processes which occur in rough were fractionated by sucrose density gradient endoplasmic reticulum (42). However, the nature centrifugation (52). We could confirm their of proteins synthesized by outer membrane-bound observation. On the other hand, GDH and ribosomes in yeast cells has not yet been elucidated. MDH were distributed in lower sucrose density As GDH in liver cells is made on the membrane portions than NADPH-cytochrome c reductase. bound ribosomes of rough endoplasmic reticulum, We also carried out immunoadsorption of micro Butow's appealing hypothesis does not seem to somes to Sepharose-bound antibody (35) and found be suitable for liver cells. that both MDH and GDH were adsorbed to the The facts that GDH was inhibited by antibody gel to the same extent even when AMG-Sepharose in intact microsomes and was released from was used. From this experiment, it may be microsomal membranes with KCl suggested its concluded that GDH and MDH are distributed outside location in microsomal vesicles. MDH, on the outside of the same microsomal vesicles. another matrix enzyme (43), was also present As the adsorption of GDH and MDH did not go in microsomes (44) and released by the same pro higher than 50%, even when the adsorption was cedure. NADPH-cytochrome c reductase which repeated using fresh AMG-Sepharose, it was is tightly bound on the outside of microsomal likely that adsorption was specific to the microsomal vesicles (45-47), and sulfite-cytochrome c reductase vesicles in which GDH and MDH were concen which is distributed on the luminal surface of trated. These results suggest the existence of microsomes (48) were not released from the specific "microparticles" in the microsomal fraction microsomes by KCl treatment. Sulfite-cytochrome and the microparticles are likely to participate c reductase is a representative of a group of enzymes in the intracellular transport of enzymes to mito which are present in the intermembrane space of chondria. mitochondria (49). The different intramicrosomal The precursor-product relationship between localization of GDH and sulfite-cytochrome c microsomal and mitochondrial GDHs confirmed reductase suggests that the compartmentation of that the enzyme activity in microsomes was not mitochondrial enzymes is determined at the step due to the adsorption of leaked enzyme from of their biosynthesis. mitochondria. We may visualize from these Blobel et al. (50) have recently proposed the observations the synthesis and intracellular trans "signal peptide" hypothesis that secretory proteins port of GDH as follows. GDH is made on are discharged into the cisterna of endoplasmic membrane-bound ribosomes and discharged on reticulum owing to the presence of short extra the outer surface of endoplasmic reticulum around sequences of hydrophobic amino acid residues the ribosomes. Subsequently, GDH on rough at their amino terminals. The outside location endoplasmic reticulum is transported to some of GDH and the inside location of sulfite-cyto specific portion of smooth endoplasmic reticulum chrome c reductase in microsomal vesicles may to be further transported into mitochondria in be related to the properties of the amino terminal about six hours. The mechanism of intracellular portions of their nascent peptides. transport of GDH is not yet known, but some

Vol. 82, No. 5, 1977 1416 K . KAWAJIRI, T. HARANO, and T. OMURA experimental data show that post translational 24. Omura, T. & Takesue, S. (1970) J. Biochem. 67, 249 modifications of enzyme molecules are likely to 25. Takesue, S. & Omura, T. (1970) J. Biochem. 67, 259 be involved in their intracellular transport from 26. Akao, T. & Omura, T. (1972) J. Biochem. 72,1245 rough and smooth microsomes to mitochondria 27. Cohen, H.J. & Fridovich, I. (1971) J. Biol. Chem. (53). 246,367 28. Arnold, H. & Maier, K.P. (1971) Biochem. Biophys. Acta 251, 133 REFERENCES 29. Fiske, C.H. & SubbaRow, Y. (1925) J. Biol. Chem. 1. Strecker, H.J. (1953) Arch. Biochem. Biophys. 46,128 66, 375 2. Ebner, E., Mennucci, L., & Schatz, G. (1973) J. Biol. 30. Beaufay, H., Amar-Costesec, A., Feytmans, E., Chem. 248, 5360 Thines-Sempoux, D., Wibo, M., & Berthet, J. (1974) 3. Ebner, E., Mason, T., & Schatz, G. (1973) J. Biol. J. Cell. Biol. 61, 188 Chem. 248, 5369 31. Lowry, O.H., Rosebrough, N.J., Farr, A.L., & 4. Ebner, E. & Schatz, G. (1973) J. Biol. Chem. 248, Randall, R.J. (1951) J. Cell. Biol. 193, 265 5379 32. Mejbaum, W. (1939) Z. Physiol. Chem. 258,117 5. Tzagoloff, A. & Meagher, P. (1972) J. Biol. Chem. 33. Allen, R.J.L. (1940) Biochem. J.34, 858 247, 594 34. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 6. Tzagoloff, A., Akai, A., & Sierra, M.F. (1972) 4406 J. Biol. Chem. 247, 6511 35. Kawajiri, K., Ito, A., & Omura, T. (1977) J. Bio 7. Rubin, M.S. & Tzagoloff, A. (1973) J. Biol. Chem. chem. 81, 779 248, 4275 36. Mason, T.L. & Schatz, G. (1973) J. Biol. Chem. 8. Okada, Y. & Omura, T. (In preparation) 248, 1355 9. Gonzalez-Cadavid, N.F. & Saez de Cordova, C. 37. Tzagoloff, A., Rubin, M.S., & Sierra, M.F. (1973) (1974) Biochem. J. 140, 157 Biochem. Biophys. Acta 301, 71 10. Roodyn, D.B., Reis, P.J., & Work, T.S. (1961) 38. Weiss, H. (1972) Eur, J. Biochem. 39, 469 Biochem. J. 80, 9 39. Hawley, E.S. & Greenwalt, J.W., (1970) J. Biol. 11. Truman, D.E.S. & Korner, A. (1962) Biochem. J. Chem. 245, 3574 83, 588 40. Schweyen, R. & Kaudewitz, F. (1970) Biochem. 12. Beattie, D.S., Basford, R.E., & Koritz, S.S. (1966) Biophys. Res. Commun. 38, 728 Biochemistry 5, 926 41. Rollston, F.S. (1974) Biochemistry 3, 91 13. Kellems, R.E. & Butow, R.A. (1972) J. Biol. Chem. 42. Redman, C.M. & Sabatini, D.D. (1966) Proc. Natl. 247, 8043 Acad. Sci. U.S. 56, 608 14. Kellems, R.E., Allison, V.F., & Butow, R.A. (1974) 43. Brdiczka, D., Pette, D., Brunner, G., & Miller, F. J. Biol. Chem. 249, 3297 (1968) Eur. J. Biochem. 5, 294 15. Kellems, R.E. & Butow, R.A. (1974) J. Biol. Chem. 44. Harano, T., Kawajiri, K., & Omura, T. (1976) 249, 3304 Seikagaku (in Japanese) 48, 660 16. Kellems, R.E., Allison, V.F., & Butow, R.A. (1975) 45. Ito, A. & Sato, R. (1969) J. Cell. Biol. 40,179 J. Cell. Biol. 65, 1 46. Kuriyama, Y., Omura, T., Siekevitz, P., & Palade, 17. Porath, J., Axen, R., & Ernback, S. (1967) Nature G.E. (1969) J. Biol. Chem. 244, 2017 (London) 215, 1491 47. Morimoto, T., Matsuura, S., Sasaki, S., Tashiro, Y., 18. Nisonoff, A., Wissler, F.C., Litman, L.N., & & Omura, T. (1976) J. Cell. Biol. 68,189 Worenley, D.L. (1960) Arch. Biochem. Biophys. 89, 48. Ito, A., Kuwahara, T., Mitsunari, Y., & Omura, T. 230 (1977) J. Biochem. 81,1531 19. Utsumi, S. & Karush, F. (1965) Biochemistry 4,1766 49. Ito, A. (1971) J. Biochem. 70, 1061 20. Gribnau, A.A.M., Schoenmakers, J.G.G., & 50. Blobel, G. & Dobberstein, B. (1975) J. Cell. Biol. 67, Bloemendal, H. (1969) Arch. Biochem. Biophys. 130, 835, 852 48 51. Eriksson, L.C. & Dallner, G. (1971) FEBS Lett. 19, 21. Porter, R.R. (1959) Biochem. J. 73, 119 163 22. Sonoda, S. & Schlamowitz, M. (1970) Immuno 52. Kuwahara, T. & Ito, A. (1975) Seikagaku (in Japa chemistry 7, 885 nese) 47, 662 23. Omura, T. & Sato, R. (1964) J. Biol. Chem. 239, 53. Kawajiri, K., Harano, T., & Omura, T. (1977) 2370, 2379 J. Biochem. 82, 1417

J. Biochem.