Identification, Purification, and Characterization of the Rat Liver Golgi Membrane ATP Transporter*

Luigi PuglielliDagger §, Elisabet C. Mandon, and Carlos B. HirschbergDagger

From the Dagger  Department of Molecular and Cell Biology, Boston University Goldman School of Dental Medicine, Boston, Massachusetts 02118 and  Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland.

We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An ~70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Post-translational modifications of proteins, occurring in the lumen of the Golgi apparatus, include glycosylation, sulfation, and phosphorylation. Although the significance and general mechanistic features of the first two reactions are well understood, very little is known about the latter one. Secreted proteins such as caseins (1, 2) and vitellogenin (3, 4) and integral Golgi membrane proteins (5) have been shown to undergo phosphorylation in the Golgi lumen. The occurrence of phosphorylated proteoglycans, including heparan sulfate, chondroitin sulfate, and proteodermatan sulfate (6-8), has also been reported. In these macromolecules, the phosphate is attached to the protein core and, in some cases, to xylose (7). Although it has been suggested that phosphorylation can contribute to maintaining the stability of the protein by protecting it against proteolytic degradation in situ (1, 2) or, in the case of proteoglycans, as a specific targeting signal (7), the significance of these post-translational modifications remains to be determined.

ATP, the phosphate donor in the above phosphorylation reactions, is synthesized mainly in the mitochondrial matrix. To be accessible to the lumen of the Golgi apparatus, where these reactions occur, it must first cross the mitochondrial membranes through a specific and well characterized transporter and then the Golgi membrane. The existence of a specific transporter in the latter membrane has been demonstrated in vitro using intact rat liver and mammary gland Golgi membrane vesicles (5). Transport was found to be temperature-dependent, saturable at micromolar concentrations of ATP and appears to be via an antiporter mechanism (9, 10), with AMP being the most likely antiporter (5). Casein phosphorylation was found to occur subsequent to transport of ATP into the lumen of rat mammary gland Golgi vesicles (5). ATP transport has also been shown to occur across the membrane of the rough endoplasmic reticulum (ER)1 (11), where it is involved in energy-requiring (reviewed in Ref. 12) and phosphorylation reactions such as of BiP (11, 13). The former include also dissociation of complexes between chaperones and correctly folded and assembled proteins in the lumen of the ER, disulfide bond formation, and protein polymerization (reviewed in Ref. 14). Recently, it has also been shown that ATP is required for protein translocation into the ER of yeast and mammalian cells (reviewed in Refs. 15 and 16).

To understand the importance of phosphorylation as a post-translational Golgi lumenal event and to analyze the possibility that regulation of ATP transport into the Golgi lumen can affect the biosynthesis and function of macromolecules being synthesized in the Golgi apparatus, we need initial knowledge of the amino acid and nucleic acid sequences of such transporter. Moreover, the existence of specific ATP transporters located in the membranes of three different intracellular organelles, mitochondria, Golgi apparatus, and ER, raises the question of how these proteins with the same function are localized in different organelles and whether or not they share common structural features.

Here we used a reconstituted phosphatidylcholine proteoliposome system (17) to monitor the purification of the ATP transport activity from a rat liver Golgi membrane preparation. Column chromatography and photoaffinity radiolabeling followed by SDS-PAGE electrophoresis were used to identify a 60-kDa protein as the ATP transporter. Proteoliposomes containing this protein were active in ATP but not in nucleotide sugars or PAPS transport; a similar apparent Km of ATP transport than previously reported for intact Golgi vesicles was determined. Finally, native functional size determination on a glycerol gradient suggested that the ATP transporter exists as a homodimer in the membrane of the Golgi apparatus.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials

Frozen rat livers were purchased from Pel-Freez Biologicals. [2,8-3H]ATP (15-30 Ci/mmol) was purchased from American Radiolabeled Chemicals, Inc., [125I]NaI (350-600 mCi/ml) was from Amersham Pharmacia Biotech, and 8-azido[gamma -32P]ATP (2-15 Ci/mmol) was from ICN Pharmaceuticals, Inc. Extracti-Gel G was purchased from Pierce. All other chemicals were obtained from Sigma.

Methods

Purification of the Rat Liver Golgi Membrane ATP Transporter All the operations described below were performed at 4 °C.

Step 1: Detergent Extraction-- A crude Golgi fraction was prepared from 6 kg of frozen rat livers according to the procedure of Leelavathi et al. (18). This fraction was resuspended in 10 mM Tris·HCl, pH 7.2, 1 mM MgCl2, 1 mM dithiothreitol, 0.3 mM sucrose, 20% glycerol (v/v), 0.3% Triton X-100 (v/v) with protease inhibitors (0.5 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 0.1 µg/ml pepstatin A, 0.1 µg/ml chymostatin, 0.1 µg/ml antipain, 0.1 µg/ml leupeptin, and 1 µg/ml aprotinin). The suspension was stirred for 45 min at 4 °C and centrifuged at 100,000 × g for 45 min. The supernatant solution was discarded, and the pellet was resuspended in the above buffer containing a final concentration of 1.1% Triton X-100. The mixture was stirred and centrifuged again as described above. The supernatant was decanted, yielding a Triton X-100 extract that was saved. The pellet was resuspended, and the extraction was repeated as described above. Both detergent extracts were combined together and adjusted to a final concentration of 0.5% Triton X-100 and 0.2 M NaCl.

Step 2: First DEAE-Sephacel Column-- The above Triton X-100 extract was applied to three DEAE-Sephacel columns (Sigma; 25 cm ×5 cm each) equilibrated in buffer A (10 mM Tris·HCl, pH 7.0, 1 mM MgCl2, 1 mM dithiothreitol, 1% glycerol, 0.5% Triton X-100) containing 0.2 M NaCl. Elution was with six column-volumes of equilibration buffer followed by four column-volumes of buffer A containing 0.7 M NaCl. The salt-eluted material containing the ATP transport activity was then concentrated and diluted to 0.3 M NaCl final concentration by use of a Minitan ultrafiltration system (Millipore).

Step 3: Blue-Sepharose Column-- The above fraction was applied to three Blue-Sepharose columns (Amicon; 20 cm × 5 cm, each) equilibrated in buffer A containing 0.3 M NaCl. The ATP transport activity eluted in the flow-through.

Step 4: Second DEAE-Sephacel Column-- The flow-through from the Blue-Sepharose column was diluted to 0.1 M NaCl with buffer A and applied to 8 DEAE-Sephacel columns (Sigma; 23 cm ×2.7 cm each) equilibrated in buffer A containing 0.1 M NaCl. The ATP transport activity eluted in the flow-through.

Step 5: Carboxymethylcellulose Column-- The second DEAE-Sephacel flow-through was applied to 12 carboxymethylcellulose columns (Amersham Pharmacia Biotech; 23 cm ×3 cm each) equilibrated in buffer A containing 0.1 M NaCl. After washing with four column-volumes of the equilibration buffer, elution was achieved with a linear gradient of 0.1-1.5 M NaCl and 20-1% glycerol. Fractions of 16 ml each were collected. Transport activity was eluted at approximately 0.6 M (fractions 12-15). The active fractions were pooled, desalted, and adjusted up to a final concentration of 10% glycerol and 0.5% Triton.

Step 6: 3',5'-ADP-Agarose Column-- The above fraction was applied to 19 3',5'-ADP-agarose columns (Sigma; 5 cm ×1.5 cm each) equilibrated in buffer A containing 10% glycerol. After washing with four column-volumes of the equilibration buffer, elution was achieved with a linear gradient of 0-1.5 M NaCl and 10-1% glycerol. Fractions of 2.5 ml each were collected, and the transport activity was eluted at approximately 0.25 M (fractions 5-15).

Glycerol Gradient The apparent functional mass of the ATP transporter was estimated by analytical ultracentrifugation using an 8-30% glycerol gradient in buffer A. The active fraction obtained from purification Step 6 was concentrated and dialyzed against buffer A in Centricon filters (Amicon) to obtain a final glycerol concentration of 8%. The 10-ml glycerol gradient was equilibrated at 4 °C for 17 h before loading of the sample (0.5 ml) and then centrifuged in a SW 50 rotor at 46,000 rpm for 40 h at 4 °C. Fractions of 0.35 ml were collected. beta -Amylase (200 kDa), alcohol dehydrogenase (150 kDa), beta -galactosidase (120 kDa), phosphorylase B (100 kDa), tumor necrosis factor alpha -convertase (80 kDa), and bovine serum albumin (66 kDa) were used as internal molecular markers.

Photoaffinity Radiolabeling with 8-Azido[gamma -32P]ATP All the following experiments were performed in a dark room in the presence of a filtered safe-light. Fractions to be photolabeled were incubated with 8-azido[gamma -32P]ATP (0.2 µM final concentration) at 0 °C for 1 min in 25 µl of buffer A. The mixture was irradiated on ice for 1 min in a Stratalinker UV 2400 oven (Stratagene; 5 cm of distance, maximum energy), and the reaction was stopped by the addition of loading buffer. Samples were then immediately subjected to 10% SDS/PAGE, and the autoradiography of the dried gel was done at -80 °C on Kodak film.

Isolation and Topography of Rat Liver Golgi Vesicles For the characterization of the ATP transporter, rat liver Golgi vesicles were isolated as described (18) and resuspended in cryoprotective buffer (19). Sialyltransferase activity was enriched ~50-fold over crude homogenate. Approximately 90% of the vesicles were sealed and of the same membrane topographical orientation as in vivo (20).

Transport Assay Transport of solutes into intact rat liver Golgi vesicles was assayed as described before (21). To follow the transporter purification, the ATP transport activity was reconstituted in phosphatidylcholine liposomes (17, 22, 23) and incubated in the presence of [3H]ATP (4 µM; 400 cpm/pmol) for 5 min at 30 °C. The reaction mixture was then applied to a 3-ml Dowex 1 × 2-100 column (Sigma) as described previously (22, 23). Fractions of 300 µl were collected, and the radioactivity was determined by liquid scintillation spectrometry.

Protein Visualization The purity of the various fractions active and inactive in ATP transport activity was determined by SDS/PAGE. Visualization was done by Coomassie Blue/silver nitrate staining (OWL Separation System) or by labeling proteins with 300 µCi of Na125I and chloramine T (22, 23). Protein was quantified using the BCA protein assay kit (Pierce).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Purification of the ATP Transporter-- The rat liver Golgi membrane ATP transporter was purified ~70,000 over the crude Golgi membrane preparation with a yield of 2% (Table I). To monitor the purification through the different purification steps, membrane proteins were reconstituted into phosphatidylcholine liposomes by freeze-thawing and then assayed for their ability to translocate radiolabeled ATP in vitro. The purity of the ATP transporter during the purification was determined by SDS/PAGE (Fig. 1). We began the purification with a crude Golgi membrane preparation; the ATP transport activity was extracted after a two-step solubilization with Triton X-100. In the first step, Golgi membranes were treated with a low concentration of Triton X-100 (0.3%, v/v). These conditions removed the peripheral membrane proteins and did not result in a significant loss of transport activity or in its extraction from the membrane. In the second step, a higher concentration of Triton X-100 was used (1.1%, v/v) to almost completely solubilize the membrane proteins. Approximately 85% of the total ATP transporter activity from the Golgi membrane preparation could be solubilized under these conditions, with a 15-fold purification over the crude Golgi membrane preparation.

                              
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Table I
Purification of the Golgi membrane ATP transporter
Membrane proteins were reconstituted in egg yolk phosphatidylcholine liposomes by freeze-thawing and then assayed for their ability to translocate radiolabeled ATP in vitro, as described under "Experimental Procedures."


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Fig. 1.   SDS-PAGE of the different chromatographic steps of the ATP transporter purification. Lane 1, Triton X-100 extract; lane 2, first DEAE-Sephacel active fraction; lane 3, Blue-Sepharose active fraction; lane 4, second DEAE-Sephacel active fraction; lane 5, carboxymethylcellulose active fraction (fraction 13 of Fig. 2); lane 6, carboxymethylcellulose inactive fraction (fraction 7 of Fig. 2); lane 7, 3'-5'-ADP-agarose active fraction (fraction 9 of Fig. 3); lane 8, 3'-5'-ADP-agarose inactive fraction (flow-through); lane 9, glycerol gradient active fraction (fraction 12 of Fig. 4); lane 10, glycerol gradient inactive fraction (fraction 2 of Fig. 4). Lanes 1-6 and 8 were visualized with Coomassie-silver nitrate staining, whereas lanes 7, 9, and 10 were visualized by autoradiography after radioiodination.

The Triton X-100 extract was then applied to a combination of conventional ion exchange, dye color, and affinity chomatography columns. Details of the different chromatographic steps are given under "Experimental Procedures," and the results of each step are given in Table I.

The Triton X-100 extract (Fig. 1, lane 1) was loaded onto a first DEAE-Sephacel column followed by elution with 0.7 M NaCl in buffer A. 72% of the transport activity was recovered with a 130-fold purification over the crude Golgi preparation (Fig. 1, lane 2). In the next two chromatographic steps, the Blue-Sepharose and the second DEAE-Sephacel column, the transport activity was found in the flow-through (negative columns). These two negative columns combined together, resulted in the binding of most of the applied proteins but not the ATP transport activity (Fig. 1, lanes 3 and 4). 41% of the initial activity was recovered after these two steps with a 2,700-fold overall purification. The active fraction obtained from the second DEAE-Sephacel was then loaded onto a carboxymethylcellulose column and eluted with a linear gradient of 0.1-1.5 M NaCl and 20-1% glycerol (Fig. 2) with a 30,000-fold overall purification (Fig. 1, lanes 5 and 6). In the next step we used a 3'-5'-ADP-agarose column, which provided an important and substantial purification (Fig. 1, lanes 7 and 8). The transport activity was eluted with a linear gradient of 0-1.5 M NaCl and 10-1% glycerol (Fig. 3). This strategy resulted in a 60,000-fold overall purification with a recovery of 8% of the initial transport activity. In order not to use high volumes of this active fraction, small aliquots of the sample were subjected to radioiodination with chloramine T before electrophoresis and visualization by autoradiography. The SDS-gel profile of the active fraction (Fig. 1, lane 7) showed two protein bands of 60- and 58-kDa. These were not visualized in fractions inactive for ATP transport activity (Fig. 1, lane 8).


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Fig. 2.   Elution profile of the ATP transport activity from the carboxymethylcellulose column. Elution was with a linear gradient of 0.1-1.5 M NaCl and 20-1% glycerol. Fractions of 16 ml were collected, and aliquots of 100 µl were used to assay ATP transport activity as described under "Experimental Procedures."


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Fig. 3.   Elution profile of the ATP transport activity from the 3'-5'-ADP-agarose column. Elution was with a linear gradient of 0-1.5 M NaCl and 10-1% glycerol. Fractions of 2.5 ml were collected, and aliquots of 100 µl were used to assay ATP transport activity, as described under "Experimental Procedures."

Glycerol Gradient-- A glycerol gradient was used as a last step of purification and to estimate the functional size of the ATP transporter. The rationale for this was based on the fact that other Golgi nucleotide sugar and nucleotide sulfate transporters appear to be homodimers in the membrane (9, 10) and, when solubilized in the presence of 0.5% Triton X-100, also behave as dimers (22, 23). The pooled active fraction from the 3'-5'-ADP-agarose column was loaded on top of a 8-30% glycerol gradient and centrifuged for 40 h, as described under "Experimental Procedures." Fig. 4A shows the profile of the transporter activity throughout the gradient; a peak in the 120-kDa area, corresponding to the native protein, was observed with a single protein band of 60 kDa in the denaturating gel (Fig. 1, lane 9, and Fig. 4B), which correlated with the ATP transport activity (Fig. 4). This latter strategy gave a 70,000-fold overall purification and a recovery of 2% of the initial transport activity.


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Fig. 4.   Glycerol gradient sedimentation of the ATP transport activity and SDS-PAGE profile. An active fraction from purification Step 6 was loaded onto an 8-30% glycerol gradient and centrifuged as described under "Experimental Procedures." Fractions of 0.35 ml were collected and assayed for the ATP transport activity. A, profile of the transport activity throughout the gradient. The numbers on the top indicate the sedimentation position of beta -amylase (200 kDa), alcohol dehydrogenase (150 kDa), beta -galactosidase (120 kDa), phosphorylase B (100 kDa), tumor necrosis factor alpha -convertase (80 kDa), and bovine serum albumin (66 kDa). B, aliquots of active and inactive fractions were radioiodinated before SDS-PAGE and then subjected to autoradiography.

Photoaffinity Radiolabeling with 8-Azido[gamma -32P]ATP-- Photoaffinity radiolabeling was used as an independent criterion to demonstrate that the transport activity is a protein of 60 kDa. We reconstituted into proteoliposomes active fractions from the glycerol gradient (see Fig. 1, lane 9) and inactive fractions from the carboxymethylcellulose (see Fig. 1, lane 6); these were then subjected to photolabeling with 8-azido[gamma -32P]ATP, an azido derivative of ATP. Only fractions active in ATP transport showed a radiolabeled protein band of 60 kDa (Fig. 5, lane 1), whereas inactive fractions did not (Fig. 5, lane 2). Neither UV irradiation, used without the photoprobe, nor the photoprobe by itself, without UV irradiation, resulted in photolabeling of protein bands (results not shown).


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Fig. 5.   SDS-PAGE of proteoliposomes subjected to photolabeling with 8-azido-[gamma -32P]ATP. Proteoliposomes were prepared as described under "Experimental Procedures." 8-Azido[gamma -32P]ATP was always used at 0.2 µM final concentration. Photolabeling was performed at 0 °C for 1 min (5 cm, maximum energy). Lane 1, proteoliposomes from the active fraction number 12 of the glycerol gradient (see Fig. 1, lane 9); lane 2, proteoliposomes from the inactive fraction number 7 of the carboxymethylcellulose (see Fig. 1, lane 6).

Characterization of the ATP Transporter-- To characterize the ATP transporter, we reconstituted the highly purified 3'-5'-ADP-agarose fraction (Fig. 1, lane 7) into phosphatidylcholine liposomes. Transport of ATP into proteoliposomes was saturable with an apparent Km of 3.3 µM (Fig. 6), very similar to that of intact Golgi vesicles (1.3 µM; Fig. 6, inset). The same fraction was inactive in transport of PAPS, CMP-sialic acid, and UDP-N-acetylgalactosamine (Table II).


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Fig. 6.   Rate of ATP transport into proteoliposomes and intact Golgi vesicles (inset). An active fraction from the 3'-5'-ADP-agarose column (fraction number 9 from Fig. 3) was reconstituted into proteoliposomes and assayed for ATP transport. Transport of ATP into intact Golgi vesicles (inset) was assayed as described before (5).

                              
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Table II
Substrate specificity of the 3'-5'-ADP-agarose column eluate
Aliquots of fraction number 9 from the 3'-5'-ADP-agarose column were reconstituted into phosphatidylcholine liposomes as described under "Experimental Procedures" and assayed for transport of ATP, PAPS, CMP-sialic acid, and UDP-N-acetylgalactosamine. The results are average of two independent determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have identified, purified, and characterized the ATP transport activity from rat liver Golgi membranes. The transporter showed an apparent molecular mass of 60 kDa, and its identity was confirmed by functional reconstitution of the purified protein into liposomes as well as photoaffinity labeling.

To purify the ATP transporter protein by column chromatography to apparent homogeneity, a ~70,000-fold purification was required. This fold of purification was expected because a similar apparent fold was required for other low-abundance Golgi membrane proteins such as the UDP-GalNAc transporter (23), the PAPS transporter (22), and the heparan sulfate N-deacetylase/N-sulfotransferase (24).

After glycerol gradient ultracentrifugation, the transporter migrated in the 120-kDa area, twice its apparent molecular mass as determined by reducing gel electrophoresis, suggesting that the ATP transporter is functional as a homodimer in Golgi membranes. These results are consistent with previous reports showing that some nucleotide derivative transporters are arranged in the Golgi membrane as homodimers (9, 10) and with analogous results obtained by us with the PAPS (22) and UDP-GalNAc transporters (23). Under the exact conditions used in this work, the PAPS transporter, a 75-kDa protein that has been shown to oligomerize as a homodimer (22), migrated in the 150-kDa area of a glycerol gradient, whereas the UDP-GalNAc transporter, a 43-kDa protein (23), migrated in the 80-90-kDa area (23).

In addition to the results obtained after column chromatography and glycerol gradient ultracentrifugation (see Figs. 1 and 4), independent evidence suggesting that the 60 kDa is indeed the ATP transporter was obtained by functional reconstitution of the transporter into proteoliposomes and photoaffinity radiolabeling using 8-azido[gamma -32P]ATP, an azido anilide derivative of ATP. When reconstituted into proteoliposomes, the highly purified transporter was active in ATP transport, and the transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles, suggesting that the two activities are identical.

The possibility that the 60-kDa protein we purified as the Golgi membrane ATP transporter is instead the ATP transporter from a different membrane, i.e. mitochondria or the ER, is very unlikely because the mitochondrial ATP transporter has a different molecular mass, ~35 kDa (25), and the one from the ER is not functional when reconstituted into proteoliposomes by the freeze-thaw procedure as used in this work (Ref. 26; see "Experimental Procedures").

Although it has been clearly shown that phosphorylation is one of the post-translational modifications that both secreted and integral Golgi membrane proteins and proteoglycans undergo during their transit through the Golgi apparatus (1-8), its functional importance is largely unknown. If indeed, as it has been suggested, it contributes to maintaining the stability of the protein backbone from proteolytic degradation in situ (1, 2) or serves as a specific targeting signal, as in the case of proteoglycans (7), remains to be determined. Understanding the importance of phosphorylation as a post-translational event and evaluation of how the possible regulation of ATP transport into the Golgi lumen can affect the biosynthesis/modification and function of macromolecules represents a major biological question. The purification of the ATP transporter constitutes an important step toward this direction. It will enable us to obtain the peptide sequence of the transporter, and from this, it will allow us to proceed toward its cloning. This in turn will enable us to study how the transporter is arranged in the membrane, if it is structurally related to the ATP transporter from mitochondria and ER, and whether its expression can regulate the post-translational modifications of the above macromolecules.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM34396 (to C. B .H.) and the Italian Consiglio Nazionale delle Ricerche (Contributo di Soggiorno to L. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a Human Frontier Science Program Organization (HFSPO) long term fellowship.

parallel To whom correspondence should be addressed: Dept. of Molecular and Cell Biology (W-200), Boston University Goldman School of Dental Medicine, 715 Albany St., Boston, MA 02118-2392. Tel.: 617-414-1040; Fax: 617-414-1041; E-mail: chirschb{at}bu.edu.

    ABBREVIATIONS

The abbreviations used are: ER, endoplasmic reticulum; PAPS, adenosine 3'-phosphate 5'-phosphosulfate; PAGE, polyacrylamide gel electrophoresis.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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