Identification of a Glycoprotein from Rat Liver Mitochondrial Inner Membrane and Demonstration of Its Origin in the Endoplasmic Reticulum*

Employing antisera against various subfractions of rat liver mitochondria (mitoplast, inner membrane, intermembrane, and matrix) as well as metabolically radiolabeled BRL-3A rat liver cells, we undertook a search for the presence of glycoproteins in this major cellular compartment for which little information in regard to glycoconjugates was available. Subsequent to [35S]methionine labeling of BRL-3A cells, a peptide:N-glycosidase-sensitive protein (45 kDa) was observed by SDS-polyacrylamide gel electrophoresis of the inner membrane immunoprecipitate, which was reduced to a molecular mass of 42 kDa by this enzyme. The 45-kDa protein was readily labeled with [2-3H]mannose, and indeed the radioactivity of the inner membrane immunoprecipitate was almost exclusively present in this component. Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) or F1F0-ATPase (complex V) also precipitated a 45-kDa protein from BRL-3A cell lysates as the predominant mannose-radiolabeled constituent. Endo-β-N-acetylglucosaminidase completely removed the radiolabel from this glycoprotein, and the released oligosaccharides were of the partially trimmed polymannose type (Glc1Man9GlcNAc to Man8GlcNAc). Cycloheximide as well as tunicamycin resulted in total inhibition of radiolabeling of the inner membrane glycoprotein, and moreover, pulse-chase studies employing metrizamide density gradient centrifugation demonstrated that the glycoprotein was initially present in the endoplasmic reticulum (ER) and subsequently appeared in a mitochondrial location. Early movement of the glycoprotein to the mitochondria after synthesis in the ER was also evident from the limited processing undergone by its N-linked oligosaccharides; this stood in contrast to lysosomal glycoproteins in which we noted extensive conversion to complex oligosaccharides. Our findings suggest that the 45-kDa glycoprotein migrates from ER to mitochondria by the previously observed contact sites between the two organelles. Furthermore, the presence of this glycoprotein in at least two major mitochondrial multienzyme complexes would be consistent with a role in mitochondrial translocations.

Although it has been widely recognized for some time that many of the macromolecular components residing in or traversing the ER 1 -Golgi system on their way to the cell surface or lysosomes are glycoprotein in nature (1), well documented information as to the presence of carbohydrate-containing proteins in other compartments of the cell has only recently emerged. Indeed, a number of reports have clearly shown that in contrast to previously held beliefs, glycosylated proteins are present in the nucleus as well as the cytoplasm (for a review, see Ref. 2). On the other hand, attempts to determine if glycoconjugates are present in mitochondria have been less convincing, and the past literature in this area consists primarily of reports utilizing carbohydrate staining and lectin binding procedures (3)(4)(5)(6)(7).
This void in information prompted us in the present study to make an intensive effort to determine if glycoproteins are indeed present in mitochondria and if so to elucidate their localization within the confines of this organelle as well as to define the nature of their carbohydrate units and the site of their formation.
Employing rat liver mitochondrial subfractions as well as the BRL-3A buffalo rat liver cell line, we have by metabolic radiolabeling and immunochemical approaches been able to demonstrate that a major 45-kDa glycoprotein bearing N-linked polymannose carbohydrate is a component of the inner mitochondrial membrane and that it is moreover associated with at least two mitochondrial enzyme complexes. Furthermore, pulse-chase experiments and studies with inhibitors indicated that this glycoprotein is synthesized in the ER and subsequently is translocated to the mitochondria.

Preparation of Rat Liver Mitoplasts and Subfractions-Mitochondria
were isolated from fasted male rats (150 -200 g, CD strain, Taconic, Inc.) by an established procedure (8,9) with only slight modifications. Briefly, this involved application of the crude mitochondrial pellet, suspended (20 mg of protein/ml) in a buffer consisting of 70 mM sucrose, 220 mM mannitol, and 2 mM HEPES, pH 7.4, to a 34-ml continuous sucrose gradient (30 -60% (w/v)) in 0.4 mM Tris/HCl, pH 7.5. Subsequent to a centrifugation at 104,000 ϫ g for 24 h, the major mitochondrial band, located at the 49% (w/v) sucrose region on the basis of marker enzyme assays, was harvested by centrifugation after a 20-fold dilution with the sucrose-mannitol, pH 7.4, buffer.
The purified mitochondria were treated with digitonin for 15 min at 0°C by the procedure of Schnaitman and Greenawalt (10) and Chan et al. (11), as summarized by Greenawalt (9), to yield mitoplasts, which were collected by centrifugation and washed with buffer at 10,000 ϫ g for 10 min. Centrifugation of the mitoplast supernatant (144,000 ϫ g, 60 min) yielded intermembrane proteins in the soluble fraction. The mitoplasts were subsequently incubated at 0°C for 15 min in Lubrol WX to yield inner membranes, which were obtained by centrifugation at 144,000 ϫ g for 60 min after a 3-fold dilution with buffer. The supernatant from this centrifugation was considered to represent matrix proteins. The purity of the mitoplasts and the mitochondria from which they were prepared was ascertained by electron microscopy through the courtesy of the Electron Microscopy Core of the Joslin Diabetes Center.
Preparation of Rat Liver Subcellular Fractions-Lysosomes were isolated from a mitochondria-lysosome fraction by centrifugation on a discontinuous metrizamide gradient by the procedure of Wattiaux et al. (12) as modified by Wong et al. (13). Rough ER was prepared by the procedure of Depierre and Dallner (14), as previously employed (15), while Golgi membranes were obtained by the method of Leelavathi et al. (16), as modified by Tulsiani et al. (17).
Radiolabeling was accomplished by incubating cells, which had reached approximately 80% confluency, on a rocker platform at 37°C with either 0.75-0.95 mCi [ 35 S]methionine (1,110 Ci/mmol) or 0.6 -1.0 mCi of [2-3 H]mannose (27 Ci/mmol), both from NEN Life Science Products, in 2 ml of methionine-free or glucose-free Dulbecco's modified Eagle's medium, respectively, containing 4 mM sodium pyruvate and 2 mM glutamine. Subsequent to a pulse of specified duration, the medium was removed and replaced with 3 ml of Dulbecco's modified Eagle's medium containing either 2 mM methionine or 2 mM mannose plus 5 mM glucose for a chase of varying periods of time. In a few experiments, the pulse was carried out with 0.6 mCi of [6-3 H]glucosamine (33 Ci/mmol, NEN Life Science Products) instead of the radiolabeled mannose. The effects of cycloheximide and tunicamycin were evaluated by including these inhibitors in the medium at concentrations of 150 and 1 g/ml, respectively, for a 30-min incubation before the addition of the radioisotope; the inhibitors were kept in the medium during a 30-min pulse but omitted during a 3-h chase. In some incubations, 0.2 mM 6-Obutanoylcastanospermine (a gift of Dr. M. Kang, Merrell Dow Research Institute, Cincinnati, OH) was included in the medium during a 30-min preincubation as well as during the pulse and chase.
At the end of the incubations, the plates were washed with ice-cold PBS (0.05 M sodium phosphate buffer, pH 7.4, containing 0.15 M NaCl) containing unlabeled substrate (2 mM methionine, mannose, or glucosamine), and the cells were then harvested by centrifugation after scraping into cold PBS containing 1 mM phenylmethylsulfonyl fluoride and aprotinin (10 units/ml). Subsequently, the cells were either homogenized in preparation for density gradient centrifugation or submitted to lysis. The latter procedure was carried out at 4°C with a lysis buffer consisting of 50 mM Tris/HCl, pH 7.6, 300 mM NaCl, 0.5% (v/v) Triton X-100, 5 mM EDTA, 10 g/ml leupeptin, 10 units/ml aprotinin, 10 mM iodoacetamide, and 2 mM phenylmethylsulfonyl fluoride followed by centrifugation at 14,000 rpm in an Eppendorf microcentrifuge for 15 min.
Metrizamide Gradient Centrifugation of Subcellular Fractions-After harvesting from the culture plates, [ 3 H]mannose-labeled or unlabeled BRL-3A cells were homogenized in 5 mM Tris/HCl, pH 7.4, buffer containing 0.25 M sucrose by 12 consecutive passages through a 22gauge needle, followed by 10 passages in a tight fitting Dounce homogenizer. The postnuclear supernatants (800 ϫ g, 10 min) of the homogenates were centrifuged at 10,000 ϫ g for 20 min to obtain a pellet, which, after suspension in 1 ml of 10 mM NaHEPES, pH 7.4, buffer containing 0.25 M sucrose, was layered on a 10-ml 2.5-40% metrizamide gradient in 10 mM NaHEPES (26,27) for centrifugation in a Beckman SW40 rotor at 114,000 ϫ g for 35 min at 4°C. Successive 1-ml fractions were removed from the top of the gradient, and after dilution with 6 ml of the buffered 0.25 M sucrose, their content was pelleted by centrifugation at 20,000 ϫ g for 20 min. After the addition of 300 l of the lysis buffer, the fractions from radiolabeled cells were analyzed by immunoprecipitation followed by PAGE. For enzyme assays, the centrifuged fractions from unlabeled cells were suspended in 20 mM Tris/ HCl, pH 7.2, containing 0.25 M sucrose. The density of the fractions from the metrizamide gradient was determined from their refractive index (28).
Preparation of Antibodies-Antisera were prepared in rabbits against rat liver mitochondrial fractions including inner membrane, intermembrane, and matrix proteins, as well as mitoplasts by a program of multiple intradermal injections (29) consisting initially of 700 g of protein followed by booster doses of 250 g of protein each. Bovine heart mitochondrial NADH-ubiquinone oxidoreductase (complex I) was kindly provided by Dr. Y. Hatefi of the Scripps Clinic and Research Foundation (La Jolla, CA), and antibodies against this enzyme, as well as against rat liver ER and lysosomal proteins, were raised in rabbits in a similar manner. Rabbit antiserum against rat liver mitochondrial F 1 -ATP synthase, which is known to immunoprecipitate mitochondrial complex V (30), was a gift of Dr. C. Godinot (Université Claude Bernard de Lyon I), while rabbit anti-mouse cathepsin L serum was generously made available by Dr. G. Sahagian (Tufts University). Antiserum against rat serum albumin raised in rabbits was purchased from Cappel.
Immunoprecipitation-Antibody-coated beads were prepared by gently shaking for 2 h at room temperature 30 l of a 1:1 suspension of PBS-washed protein A-Sepharose CL-4B beads (Sigma) with 30 l of antiserum or preimmune serum, followed by washing with PBS and PBS containing 0.1% (w/v) bovine serum albumin. Aliquots of cell lysates and lysed metrizamide gradient fractions, precleared by centrifugation at 14,000 ϫ g for 15 min in an Eppendorf microcentrifuge, were then added to the coated beads and shaken for 2 h at room temperature in a total volume of 400 l of lysis buffer containing a mixture of protease inhibitors. After pelleting by centrifugation (800 ϫ g for 10 min at 4°C), the beads were washed four times with 500 l of a solution containing 50 mM Tris/HCl, pH 7.6, 300 mM NaCl, 0.1% (v/v) Triton X-100, 0.02% SDS, and protease inhibitors. Protein was then released from the washed beads by boiling for 5 min in PAGE sample buffer containing 1% SDS and 2% (v/v) 2-mercaptoethanol.
SDS-PAGE and Immunoblotting-Electrophoresis was carried out on 7-10 or 7-15% gradient polyacrylamide gels (1.5 mm thick), overlaid by a 3.5% stacking gel, in SDS according to the procedure of Laemmli (31). Radioactive components were detected by fluorography, while protein bands were visualized by the Coomassie or silver stains.
For immunological identification, the proteins were transferred onto nitrocellulose sheets (32). The sheets were washed with PBS, 0.1% bovine serum albumin followed by PBS, 0.1% bovine serum albumin containing 0.05% Tween 10, and then they were incubated for 1 h at room temperature with a 200-fold diluted normal goat serum. Subsequent to further washes with this solution, incubation of the sheets with diluted antiserum was carried out for 2 h at room temperature. Bound antibody was then detected with 125 I-labeled protein A followed by autoradiography as described previously (33).
PNGase and Endo H Digestions-Immunoprecipitated protein was released from the protein A beads by boiling for 3 min in 25 l of buffer containing 0.5% SDS and 0.1 M 2-mercaptoethanol. The solubilized protein obtained after removal of the beads by centrifugation was then incubated in a 125-l volume with either 0.6 units of PNGase (PNGase F, Oxford GlycoSystems) or 10 milliunits of endo H (Genzyme) for 30 and 48 h, respectively, at 37°C in the presence of aprotinin (10 units/ ml), phenylmethylsulfonyl fluoride (2 mM), and toluene. The composition of the buffer for the PNGase digestion was 60 mM Tris/HCl, pH 8.6, 6 mM EDTA, 1.0% (v/v) Nonidet P-40, 0.1% SDS, and 0.02 M 2-mercaptoethanol, while that for the endo H treatment was 0.2 M sodium citrate, pH 5.2, 0.1% SDS, and 0.02 M 2-mercaptoethanol. At the end of the digestions, the samples, as well as control incubations without enzyme, were examined by SDS-PAGE followed by fluorography.
Analysis of N-Linked Oligosaccharides-Glycopeptides were prepared from [2-3 H]mannose-labeled immunoprecipitated proteins by incubating the beads to which they were bound with Pronase (Calbiochem), as described previously (34). Endo H treatment of the glycopeptides released polymannose oligosaccharides, which, after passage of the digests through coupled columns of Dowex 50 (H ϩ ) and Dowex 1 (acetate), appeared in the effluent and water wash (35); the resins were eluted with 2 M pyridine acetate, pH 5.0, to recover endo H-resistant peptide-linked carbohydrate units (35). The nature of the polymannose oligosaccharides was evaluated by thin layer chromatography on plastic sheets precoated with Silica Gel 60 (0.2-mm thickness, Merck) in 1-propanol/acetic acid/water (3:3:2), and the components were visualized by fluorography as previously reported (35).
Concanavalin A Binding-Immunoprecipitated radiolabeled protein after elution from protein A beads was adjusted to an SDS concentration of 0.05% and added to 0.15 ml of packed concanavalin A-Sepharose beads (Amersham Pharmacia Biotech), which had been equilibrated with 50 mM Tris/HCl, pH 7.8, buffer containing 100 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 0.2% (v/v) Triton X-100, and 0.02% sodium azide. Subsequent to shaking for 1 h at room temperature, the supernatant was removed, and after several washes with the adsorption buffer, the bound protein was eluted sequentially with 300 mM methyl ␣-D-mannoside and by heating the beads at 100°C for 5 min in PAGE sample buffer containing 5% 2-mercaptoethanol. The unbound and bound fractions were then examined by SDS-PAGE.
Radioactivity Measurements-Liquid scintillation was carried out in Monofluor (National Diagnostics) with a Beckman LS7500 instrument. Components on electrophoretic gels and thin layer chromatographic plates were detected by fluorography at Ϫ80°C with the use of X-Omatic AR film (Eastman Kodak Co.) after treatment with ENHANCE (NEN Life Science Products) or spraying with a scintillation mixture containing 2-methylnaphthalene (36), respectively. Components on immunoblots were visualized by autoradiography.

Evaluation of the PNGase Susceptibility of Mitochondrial Proteins Labeled with [ 35 S]Methionine in BRL-3A Cells-In
order to determine whether glycoproteins are present in mitochondria and to assess their site of synthesis and nature of their carbohydrate units, we chose an approach that involves immunoprecipitation of defined components after metabolic radiolabeling of cultured cells. Examination of SDS-PAGE immunoblots of antisera prepared in rabbits against rat liver mitoplast and its various subfractions (inner membrane, intermembrane, and matrix) indicated that they provided a suitable tool for such experiments, because whereas they yielded a distinctive multibanded pattern when employed against their respective fractions, they did not react with other purified rat liver membrane fractions including ER, lysosomes, and Golgi (data not shown). Conversely, antibodies directed against rat serum albumin and cathepsin L did not react with the mitochondrial components, whereas as anticipated they bound strongly to these proteins on immunoblots of ER and lysosomal fractions, respectively.
After radiolabeling of the rat liver BRL-3A cell line with [ 35 S]methionine, immunoprecipitates obtained with antisera against mitochondrial subfractions were examined by SDS-PAGE with or without prior PNGase digestion to assess the possibility that glycoproteins are present (Fig. 1). Indeed, a comparison of the enzyme-treated and untreated samples indicated that the inner membrane contained a component of 45 kDa, which was reduced in mass to approximately 42 kDa, suggesting the release of N-linked carbohydrate. A similar PNGase-sensitive protein was evident in the mitoplast (Fig. 1), and after prolonged fluorography it could also be detected in the intermembrane fraction. Examination of the immunoprecipitate from antisera directed against the mitochondrial matrix did not reveal this protein or any other PNGase-sensitive component (data not shown).
Mannose Radiolabeling of Mitochondrial Glycoproteins-Since the studies with PNGase indicated that the inner membrane contains a relatively prominent glycoprotein, our subsequent studies focused on this mitochondrial subfraction. Indeed, incubation of BRL cells with [2-3 H]mannose to specifically label glycoproteins with N-linked oligosaccharides indicated that the inner membrane immunoprecipitate contained a major radiolabeled component with a molecular mass of approximately 45 kDa, which was predictably also present in the mitoplast but only barely perceptible in the mitochondrial in-termembrane compartment (Fig. 2). Moreover, antisera directed against mitochondrial NADH-ubiquinone oxidoreductase (complex I) and ATPase (complex V) immunoprecipitated from the lysate of [2-3 H]mannose-labeled BRL cells a 45-kDa protein as the major radioactive component (Fig. 3); aside from this predominant 45-kDa protein, minor radioactive bands could be revealed by prolonged fluorography. Since digestion of The immunoprecipitates, after elution from the protein A-Sepharose beads, were digested with (ϩ) or without (Ϫ) PNGase prior to SDS-PAGE (7-10% gradient). The components were visualized by fluorography; the designated molecular weight markers were Escherichia coli ␤-galactosidase (116,000); rabbit muscle phosphorylase (97,000); bovine serum albumin (66,000), and hen ovalbumin (45,000). Arrows indicate protein bands that differ between the control and PNGase-treated samples. The presence of the nonradiolabeled IgG heavy chain in the samples results in some distortion in the migration of the radiolabeled components in the 50-kDa region of the untreated samples, which is not apparent in the PNGase-digested proteins since the IgG chain is itself reduced in size and migrates at a faster rate. these immunoprecipitates with endo H completely released the radiolabel from the 45-kDa component, it can be inferred that the [2-3 H]mannose is present in one or two N-linked polymannose oligosaccharides (Fig. 3).
When BRL cells were radiolabeled with [6-3 H]glucosamine, the 45-kDa radiolabeled band was also seen upon SDS-PAGE as the predominant radioactive component of the mitochondrial inner membrane immunoprecipitate (data not shown).
Furthermore, when the [2-3 H]mannose labeling protocol was applied to another rat cell line (NRK-45F) a major 45-kDa radioactive component was also seen upon SDS-PAGE of the immunoprecipitate of the inner mitochondrial membrane (data not shown).
Separation of BRL-3A Subcellular Fractions by Metrizamide Density Gradient Centrifugation-In order to confirm the subcellular locale of the 45-kDa glycoprotein observed in the immunoprecipitates of the mitochondrial inner membrane and the mitochondrial enzyme complexes examined, we employed metrizamide gradient centrifugation. By this procedure, partial resolution of lysosome, mitochondria, and ER was achieved, as determined by enzyme markers, so that the peak tubes of the organelles were well separated (Fig. 4).
Since antibodies directed against ER, mitochondrial inner membrane, and lysosomal proteins immunoprecipitated in each case a distinct group of components from lysates of [2-3 H]mannose-labeled BRL cells (Fig. 5), the antibodies could be utilized to follow the distribution of these subcellularly localized glycoproteins on a metrizamide density gradient. Indeed, the distribution of the lysosomal, inner mitochondrial membrane, and ER immunoprecipitates along the gradient was very similar to that observed with the respective enzyme markers (cf. Fig. 4 and Fig. 6, left panel). When we examined the immunoprecipitates throughout the gradient by SDS-PAGE, the glycoprotein banding pattern distinctive for each subcellular fraction was observed across its respective peak, and this is exemplified by the fluorographs from the tubes showing maximal radioactivity (Fig. 6, right panel). The occurrence of a small inner mitochondrial membrane peak overlapping with that of the lysosome most likely represents the in- FIG. 4. Separation of BRL-3A subcellular fractions by metrizamide gradient centrifugation. After homogenization of BRL cells from five 100-mm plates, the suspended pellet obtained by centrifugation of the postnuclear supernatant at 10,000 ϫ g for 20 min was loaded on a 2.5-40% metrizamide gradient as described under "Experimental Procedures." Subsequent to a 35-min centrifugation (114,000 ϫ g), 1-ml fractions were collected starting from the top and analyzed for marker enzymes including ␤-N-acetylglucosaminidase (GN), NADPH-cytochrome c reductase (CR), and malate dehydrogenase (MD) to identify fractions containing lysosomes, ER, and mitochondria, respectively. Each enzyme activity is plotted as a percentage of the activity observed in the peak tube. Density was determined from the refractive index.

FIG. 5. Electrophoresis of [2-3 H]mannose-labeled glycoproteins immunoprecipitated from BRL-3A cell lysate by antisera utilized to analyze metrizamide gradient fractionation. Subsequent to incubation of BRL cells with [2-3 H]mannose (2-h pulse, 3-h chase)
, lysates of the cells were reacted with antisera against ER, mitochondria inner membrane (IM), and lysosomes (LY) as described under "Experimental Procedures." The immunoprecipitates were then submitted to SDS-PAGE (7-15% gradient), and the components were visualized by fluorography. The molecular weight markers are the same as in Fig. 1. When the lysate was reacted with the anti-inner membrane serum after a preimmunoprecipitation with antiserum against ER, the electrophoretograph of inner membrane was indistinguishable from that shown. The immunoprecipitates after elution from the protein A-Sepharose beads were submitted directly to SDS-PAGE (7-15% gradient, left panel) or digested with (ϩ) or without Endo H (Ϫ) followed by PAGE (7-10% gradient, right panel). Components were detected by fluorography; the molecular mass markers are the same as in Fig. 1. corporation of mitochondrial material into the latter by autophagy, as has been reported in liver cells (37). [2-3 H]mannose in the presence of either cycloheximide or tunicamycin in a manner described under "Experimental Procedures" resulted in each case in a total inhibition of inner mitochondrial membrane glycoprotein formation (data not shown). These findings suggested that the synthesis of these molecules takes place outside of the mitochondrial compartment and that it moreover involves an N-glycosylation mechanism characteristic of the ER.

Evaluation of the Subcellular Site for the N-Glycosylation of Mitochondrial Glycoprotein-Incubations of BRL cells with
Pulse-chase studies employing a metrizamide density gradient provided data that supported the likelihood of an ER origin of mitochondrial glycoprotein by demonstrating an early localization to this compartment. After a 30-min pulse, glycoproteins immunoprecipitated by antisera directed against mitochondrial inner membrane and NADH-ubiquinone oxidoreductase (complex I) co-centrifuged with ER constituents, whereas after a 2-h chase, the mitochondrial components were found to be concentrated in gradient fractions of higher density (Fig. 7), which is consistent with mitochondrial localization as determined by enzyme markers (Fig. 4) or immunoprecipitates after prolonged incubations (Fig. 6). Similarly, as anticipated, lysosomal glycoproteins that initially appeared in the ER region of the metrizamide gradient subsequently moved to their characteristic lighter position (Fig. 7).
Nature of the Carbohydrate Units of Mitochondrial Glycoproteins-Upon endo H digestion of glycopeptides derived from BRL cell immunoprecipitates obtained with antisera directed against mitochondrial NADH-ubiquinone oxidoreductase, ER, and lysosomes after a 30-min pulse with [2-3 H]mannose, greater than 85% of the radioactivity was released as polymannose oligosaccharides. However, subsequent to a 3-h chase, while the endo H sensitivity of the lysosomal glycopeptides was reduced to 23%, the susceptibility to cleavage of the mitochondrial and ER glycopeptides remained essentially unchanged. Thin layer chromatography of the endo H-released oligosaccharide clearly indicated that the processing of the ER and mitochondrial glycoproteins were strikingly similar and did not progress substantially beyond the Man 8 GlcNAc stage, while those of the lysosomes underwent trimming to complex carbohydrate units, which is characteristic of movement through the ER-Golgi pathway (Fig. 8). The distinctive presence of small amounts of Glc 1 Man 9 GlcNAc was observed after the 30-min pulse in the three immmunoprecipitates, suggesting a similar N-glycosylation mechanism. This was further evident from the observation that in the presence of castanospermine, Glc 3 Man 9 GlcNAc was the predominant oligosaccharide species released by endo H from glycopeptides derived from the entire mitochondrial inner membrane as well as ER immunoprecipitates (data not shown).
The presence of N-linked polymannose oligosaccharides in mitochondrial glycoprotein was also demonstrated by the find- the homogenate at 10,000 ϫ g for 20 min was applied to a metrizamide gradient as described under "Experimental Procedures." One-ml fractions were collected after a 35-min centrifugation (114,000 ϫ g), and subsequent to the addition of lysis buffer to the membranes obtained from these fractions, immunoprecipitation was carried out on aliquots with antisera against lysosomes (LY), ER, and mitochondrial inner membrane (IM). The radioactivity recovered in the immunoprecipitates is plotted as a percentage of the radioactivity observed in the peak tube, which was 7.8 ϫ 10 4 , 8.0 ϫ 10 3 , and 4.2 ϫ 10 3 dpm for lysosomes, ER, and mitochondrial inner membrane, respectively (left panel). The immunoprecipitates were examined by SDS-PAGE (7-10% gradient), and the components were visualized by fluorography. The electrophoretographs for the peak tubes are shown (right panel); other fractions for each antisera gave identical patterns but of lesser intensity.  Fig.  6 on cells pulsed without and with a subsequent chase. Fractions of 1 ml were collected, and after the addition of lysis buffer immunoprecipitation was carried out on aliquots with antisera against mitochondrial inner membrane (IM), ER, NADH-ubiquinone oxidoreductase (UBI), and lysosomes (LYS). The radioactivity in the immunoprecipitates is plotted as a percentage of radioactivity observed in the peak tube. The radioactivity in this tube after the pulse was 3.5 ϫ 10 4 , 2.6 ϫ 10 4 , 1.1 ϫ 10 4 , and 14.4 ϫ 10 4 dpm for inner membrane, ER, NADH-ubiquinone oxidoreductase, and lysosomes, respectively, while after the chase it was 1.9 ϫ 10 4 , 1.8 ϫ 10 4 , 6.6 ϫ 10 3 and 13.2 ϫ 10 4 dpm for inner membrane, ER, NADH-ubiquinone oxidoreductase, and lysosomes, respectively. The dashed line indicates the position of the peak ER fraction to facilitate comparison of the plots in the upper and lower panels.
ing that when the [ 35 S]methionine-labeled protein from an anti-NADH-ubiquinone oxidoreductase (complex I) immunoprecipitate was reacted with concanavalin A-coated beads, the 45-kDa protein was the predominant component adsorbed to the lectin, while most of the other multiple subunits of this mitochondrial complex remained unbound (Fig. 9).

DISCUSSION
The present study has provided evidence that a 45-kDa glycoprotein bearing N-linked polymannose oligosaccharide occurs as an integral component of rat liver mitochondria. Indeed, the finding that this glycoconjugate is located in the mitochondrial inner membrane in which the enzymes of the respiratory chain and energy transduction are situated suggests that it may carry out an important role. This possibility was strengthened by our observation that antibodies directed against two of the major multisubunit enzyme complexes of the inner membrane (30,38), namely NADH-ubiquinone oxidoreductase (complex I) and F 1 F 0 -ATP synthase (complex V) precipitated the 45-kDa glycoprotein.
The occurrence of an N-glycosylated protein in the interior of a subcellular compartment unrelated to the ER-Golgi system was quite surprising and focused our attention on defining the site of its synthesis. Clearly, the assembly of N-linked carbohydrate requires a considerably more complicated enzymatic machinery than that needed for the attachment of the O-linked GlcNAc residues that have been characterized in nuclear and cytoplasmic locations (2). It became evident to us from pulsechase studies that the mitochondrial N-linked oligosaccharides bore a striking resemblance to those observed on ER glycopro-teins and like the latter underwent only very limited processing (39,40), which did not extend substantially beyond the Man 8 GlcNAc stage, and were sensitive to tunicamycin inhibition. Indeed, in comparable fashion, the mitochondrial and ER glycoproteins remained in the immature endo H-susceptible state, and moreover our studies suggested that N-glycosylation of proteins from both locations was brought about by the attachment of the well characterized triglucosylated polymannose oligosaccharide (for reviews, see Refs. 41 and 42). The pulse-chase metrizamide gradient fractionation experiments indicated that the similarity between the glycoproteins from these two subcellular sites was not coincidental but that the ER was indeed the site for the N-glycosylation of mitochondrial protein. This was a reassuring finding, since alternatively it would be necessary to postulate that the mitochondria have a duplicate of the highly complicated multienzyme biosynthetic machinery that has been described for the assembly, transfer, and initial trimming of N-linked oligosaccharides in the ER (41,42). Although some reports of glycosyltransferases in mitochondria have been published (43,44), it would appear unlikely from our findings that they are involved in the in situ formation of mitochondrial glycoprotein.
Since the ER enzymatic machinery appears to be responsible for the N-glycosylation of mitochondrial glycoproteins, a novel mechanism for the transfer of protein from one compartment to the other must be invoked that is distinct from the well established pathway employed for the importation of proteins into the mitochondria directly from the cytosol (45,46). Recent studies have indicated that a translocation from ER to cytosol of polymannose oligosaccharides does occur (47) and that probably glycopeptides (48) and glycoproteins (49,50) containing such carbohydrate units can also make this passage. However, if such an event were responsible for ER-mitochondrial interchange, another translocation from cytosol to the latter compartment would have to be postulated. In the absence of experimental data, we would prefer to consider as a model the well documented transport of newly synthesized phospholipids from ER to mitochondria (51)(52)(53). It has been shown that phosphatidylserine after synthesis in the ER is imported into the mito-

FIG. 8. Thin layer chromatographic identification of the endo H-released N-linked oligosaccharides of a mitochondrial glycoprotein in comparison with ER and lysosomal constituents during pulse-chase [2-3 H]mannose radiolabeling of BRL cells.
After radiolabeling of the cells with a 30-min pulse with and without a subsequent 3-h chase, immunoprecipitation of lysates was accomplished with antisera against NADH-ubiquinone oxidoreductase (UB), ER, and lysosomes (LY). Glycopeptides prepared from the immunoprecipitates were then treated with endo H as described under "Experimental Procedures," and the oligosaccharides released by this enzyme were then applied to silica gel-coated plates for chromatography in 1-propanol/acetic acid/water (3:3:2) for 18 h. Aliquots representing an equal percentage of the samples after the pulse and pulse-chase incubations were submitted to chromatography, and the components were detected by fluorography. The migration of standard oligosaccharides is indicated to the left of the chromatography by the following designations: G 1 , Glc 1 Man 9 GlcNAc; M 9 , Man 9 GlcNAc; M 8 , Man 8 GlcNAc; M 7 , Man 7 GlcNAc; and M 6 , Man 6 GlcNAc. When the oligosaccharides released by endo H from glycopeptides derived from a total inner membrane immunoprecipitate were submitted to TLC, a pattern similar to that for NADH-ubiquinone oxidoreductase was seen. , a lysate of the cells was reacted with antiserum against the mitochondrial enzyme. The solubilized immunoprecipitate was then added to concanavalin A-Sepharose beads (ConA) and equal aliquots of the unbound (Ϫ) and bound (ϩ) protein was then submitted to SDS-PAGE (7-10% gradient) as described under "Experimental Procedures." The components were visualized by fluorography; molecular weight markers are the same as in Fig. 1. chondrial inner membrane, where it undergoes decarboxylation to form phosphatidylethanolamine (51). Most relevant to our inquiry is the finding that this translocation is mediated by a functionally distinct region of the ER, which is in direct contact with the mitochondrial membrane and can function as a "bridge" between the two compartments. It has been known for some time that regions of contact exist between ER and mitochondria (54,55), and indeed this mitochondria-associated membrane (MAM) has been isolated and shown by in vitro experiments to function as a pre-Golgi compartment of the secretory route, which does not involve cytosolic factors (52). It is of interest that in a recent report a "fusogenic" protein, believed to be involved in the import of phosphatidylserine, has been isolated from rat brain mitochondria and shown on the basis of concanavalin A binding to be a glycoprotein (56).
Our observation that the 45-kDa glycoprotein described in the present study is a component of at least two major enzyme complexes of the inner mitochondrial membrane suggests that it may play a role in mitochondrial translocations. Of relevance to this speculation are the recent reports that in yeast, TIM11, which is believed to be a component of the inner membrane import machinery (57), has been found to be identical to mammalian F 1 F 0 -ATPase subunit e (58) and is also associated with the mitochondrial NADH-cytochrome b 5 reductase (59).
Clearly, the presence of a 45-kDa N-linked polymannose bearing glycoprotein in the interior of the mitochondrial compartment invites challenging investigations into an area of glycobiology that remains largely unexplored.