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Reconstitution of adenosine 3′-phosphate 5′-phosphosulfate transporter from rat brain
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 155, No. 3, 1988
Pages 1271-1277
September 30, 1988
RECONSTITUTION OF ADENOSINE 3'-PHOSPHATE 5'-PHOSPHOSULFATE TRANSPORTER FROM RAT BRAIN M.E. Zaruba*, N.B. Schwartz§ and G.I. Tennekoon*
Department of Neurology*, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the Department of Pediatrics§, University of Chicago, Chicago, Illinois 60637 Received August 8, 1988
SUMMARY: Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the "active" sulfate donor for sulfated macromolecules, is synthesized in the cytosolle fraction of rat brains. This molecule is then translocated into the lumen of the Golgl apparatus so that it is available to the sulfotransferase enzymes. The protein responsible for the PAPS translocatlng activity has been solubillzed from vesicles enrlehed in enzyme markers for the Golgi apparatus and reconstituted into llposomes. In reconstituted liposomes translocatlng activity has a pH optimum of 7.0 and activity was increased 3-fold by divalent cations, although EDTA produced no inhibition. The affinity of the reconstituted translocator for PAPS showed a Km of 1.2 m M w i t h a Vmax of 14 pmol of PAPS translocated/mln/mg of protein. Specificity of the translocator activity was tested with a number of nucleotlde analogues and only 3',5'adenosine diphosphate was a competitive inhibitor. Inhlbitors of the mltochondrial ADP/ATP transporter and the red cell anion channel blocked transport of PAPS only at very high concentrations. © 19s8AcaaemioPress, ~nc.
Sulfation of macromolecules takes place largely in the Golgi complex (6,7,15,18,26).
For example, cerebroside sulfotransferase, the enzyme that
catalyzes the sulfation of galactocerebroside, was found to be localized to the Golgi apparatus in brain and kidney (5-7) and had a luminal orientation in trans-Golgl (22). The enzymes involved in the synthesis of the active sulfate donor adenosine 3'-phosphate 5'-phosphosulfate sulfurylase and adenosine 5'-phosphosulfate
(PAPS), ATP-
(APS)kinase, had been localized
to the cytosol in yeast (17) and Hirschberg and co-workers (2,19) have shown the presence of a translocatlng molecule for PAPS in intact Golgi vesicles from liver. For an understanding of the mechanism of regulation of sulfatlon in rat brain, it will be necessary to purify the PAPS transporter.
Abbreviations used:
A prerequisite
APS, adenosine 5'-phosphosulfate; PAPS, adenosine 3'-
phosphate 5'-phosphosulfate.
0006-291)<)88 $1.50 1271
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for purifying this "carrier" molecule is the ability to solubilize and reconstitute them in liposomes.
In this communication, we describe the
reconstitntion into liposomes of the PAPS translocating molecule and the subcellular localization of the PAPS synthesizing enzymes in rat brain.
MATERIALS AND METHODS Sodium cholate, sodium phosphate, soybean lipids, mercuric chloride, ammonium acetate, acetyltrypsin and soybean trypsin inhibitor, 4,4'diisothiocyanostilbene 2,2'-disulfonic acid and palmitoyl-CoA were obtained from Sigma. Triton X-100 was purchased from Rohm and Haas. All the nucleotides used in this study were purchased from P.L. Biochemicals. [3H]Methoxy inulin (5,000 Ci/mmol) was purchased from ICN Radiochemicals. [35S]Adenosine 3'-phosphate 5'-phosphosulfate (1.2-2.8 mCi/mmol), [35S]sodium sulfate and [32p]Na4P207 (I-60 Ci/mmol) were obtained from New England Nuclear. Microsome Vesicle Preparation. The microsome vesicles (Golgi-enriched) were prepared as described (22), from brains of i0- to 20-day-old SpragueDawley rats (Charles River, MA). Reconstitution of Vesicles. The reconstitution of microsomal protein into liposome vesicles was based on procedures previously described (1,9,10,13). Microsomal proteins (3.0 mg/ml) were solubilized for 1 h at 4°C in I0 mM sodium phosphate, I00 mM NaCI, pH 7.4 (PBS) containing 2% sodium cholate, and 0.2% crude soybean lipids. After solubilization, the protein was diluted to 1.3 mg/ml with 2% sodium cholate containing 2% soybean lipids, sonicated for 30 sec, and then dialyzed against PBS containing 10 mM sodium azide (four changes each of 50 volumes over 60 h). Transporter Assay. Transport activity was measured by adding l0 ~l of 0.2 mM [35S]PAPS (400,000) dpm) to 200 ~I of reconstituted liposomes (I-2 mg/ml of protein) to which i0 ~l of 200 mM CaCI 2 had been added (final concentration of CaCI 2, 10 mM), and incubation at 20°C or as indicated. The assay was stopped at specified times by adding I0 ~l of 200 mM HgC12 and placing the mixture on ice. The incubation mixture was then added to a 2.0ml Sephadex G-100 column equilibrated and then eluted with PBS. The 5-drop column fractions were collected and counted in 5.0 ml of liquiscint (National Diagnostics). This procedure provides good separation of the liposomeassociated [35S]PAPS from the "free" [35S]PAPS. Identification of 35S-labeled Materials. Twelve 200-~1 aliquots of reconstituted vesicles were incubated as described for the transporter assay. The vesicle fractions, separated from the "free" PAPS on the Sephadex G-100 columns, were pooled, frozen and lyophilized. The lyophillzed sample was brought to 1.0 ml with ethanol:H20, l:l (v:v) and 20-~1 aliquots were spotted (23) in duplicate on Whatman No. 3 paper with appropriate nucleotide markers and developed (ascending) for 4 h in I M ammonium acetate:ethanol (7:3, v/v), pH 7.5. The products were then visualized by UV light, cut out, extracted for 30 min with l.O ml of H20, and counted in lO ml of liquiscint (National Diagnostics). For identification of 35SO4-1abeled lipid and proteins, a 0.5-ml aliquot of the lyophilized vesicle sample in ethanol:H20, I:I (v/v) was extracted in 5 ml of chloroform:methanol, i:i (v/v) for 60 min at 4°C and then centrifuged at 400 x g for 25 mln at 4°C. The supernatant fraction was decanted and saved. The pellet was again extracted and centrifuged and the supernatant fractions were pooled, subjected to lipid extraction, and partitioned according to Folch et al. (8). The pellet remaining from the chloroform:methanol extraction was washed three times with I0 ml of 6% trichloracetic acid and 0.5% phosphotungstic acid, with centrifugation for 15 min at 4°C at 300 x g between washes. It was then solubillzed in 1.0 ml of N,C.S. tissue solubilizer (Amersham), 1272
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
decolorized with I00 ~i of 30% hydrogen peroxide overnight at 42°C, and counted by liquid scintillation counting. Sensitivity of Reconstituted Transporter to Acetyltrypsin. For each time point, 200 BI of reconstituted vesicles (0.26 mg of protein) were preincubated for specified times at 20°C with i0 ~I of acetyltrypsln (i mg/ml). Then I0 ~I of PBS containing 5 mg/ml of soybean trypsin inhibitor were added, the sample was mixed with a Vortex mixer and assayed for PAPS transporter activity by the transporter assay described above. Controls with buffer instead of acetyltrypsln were included. Enzyme Assays. Subcellular fractionation was performed as previously established in our laboratory (3). Standard definitions are used for mitochondrial, microsomal and cytosolic fractions. ATP sulfurylase was assayed by measuring the formation of [32p]ATP from adenosine 5-phosphosulfate and 32p-labeled pyrophosphate as described by Sugahara and Schwartz (4,20,21). APS kinase was assayed by measuring conversion of [35S]APS to [35S]PAPS as described by Sugahara and Schwartz (20). Protein Determination. Proteins were assayed by the method of Lowry et al. (12) with bovine serum albumin as standard.
RESULTS AND DISCUSSION Localization of Enzymes that Synthesize Adenosine 3'-Phosphate 5'Phosphosulfate.
The specific activities of ATP-sulfurylase and APS-kinase
were determined in homogenates and various subcellular fractions (Table I). Specific activities were highest in the supernatant fractions, establishing that the enzymes responsible for the synthesis of PAPS were localized in the cytosol of cells, similar to those reported for yeast.
Hence, PAPS must be
translocated into the Golgi complex so that it is available to the sulfotransferase. Reconstitution of PAPS Translocator.
We first ensured that the PAPS
translocator was present in Golgi-enriched microsomes
(data not shown).
We
next tested a variety of detergents and found that rat brain microsomes were extracted only with 2% sodium cholate or 1.5% octylglucoside.
Both
detergents extracted 60% of the total microsomal proteins, which showed identical PAPS translocating activities,
and in all subsequent experiments
the initial extraction was carried out with 2% sodium cholate and proteins were reconstituted with 0.2% soybean lipids as described under "Materials and Methods." Characterization of PAPS Translocating Activity in Reconstituted Liposomes.
Both [2-3H]methoxyinulin
and carrier-free
[35S]sulfate were
excluded from the liposomes, suggesting that the liposomes were not leaky to small molecular weight compounds.
Moreover, no translocation of [35S]PAPS
was observed in the absence of extracted protein fraction, or in the presence of other protein fractions, including bovine serum albumin, protein extracts from red cell ghosts, or proteins obtained by extraction of boiled microsomes.
The translocating activity was decreased by about 70% by
1273
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
TABLE 1.
Subcellular localization of PAPS synthesizing enzymes in rat brain
Fraction
Specific Activity APS Kinase ATP Sulfurylase cpm/mg protein/min 104
Homogenate 1000 x g supernatant i000 x g pellet (nuclei) 12,000 x g supernatant 12,000 pellet x g (mitochondria) 00,000 x g supernatant (cytosol) i00,000 x g pellet (microsomes)
1.51 1.95 1.40 4.96 1.37 8.96 0.86
1.50 1.50 0.45 3.93 0.67 5.72 0.54
Whole brains from 14-day-old Sprague-Dawley rats were removed and homogenized by use of a Dounce homogenizer. The homogenate was centrifuged at 1,000 x g for i0 min, the pellet (nuclei) was washed once, and the supernatant fractions were combined and centrifuged at 12,000 x g for 30 min. The pellet (mitochondria) was washed and the supernatant franctions were combined and centrifuged at i00,000 x g for 90 min. The supernatant fractions were designated as cytosol and the pellet constituted the microsomal fraction. Details of the assays are described in the text.
treatment with acetyltrypsin,
suggesting
that the translocating molecule is a
protein. In the reconstituted
liposomes,
the rate of translocation
of PAPS was
linear for the first 7 min, after which the rate decreased but still remained significant.
Translocation
concentration about 7.0.
activity was proportional
(8-154 Bg), temperature-dependent,
There was demonstrable
added divalent
cations,
slight stimulatory
translocating
to protein
and showed a pH optimum of activity in the absence of
and EDTA did not inhibit translocation,
effect.
Nevertheless,
but had a
divalent cations increased
the rate
of PAPS translocation by 2- to 3-fold, with calcium having the greatest stimulatory
effect.
concentrations
Transport of [35S]PAPS as a function of different
of this compound showed typical saturation kinetics and a
double reciprocal plot gave a linear relationship with a Km=l.2 mM and a Vmax=14 pmol of PAPS translocated/min/mg reconstituted
transporter
Identification counts translocated
is different
of protein
(Fig.
IA).
The K m of the
from that in intact vesicles
of 35S-labeled Material.
(6 BM).
Analysis of the [35S]PAPS
showed that about half the counts were associated with
protein and very few (about 1%) of the counts with the lipid fraction. remaining
counts were soluble in 10% trichloroacetic
soluble material [35S]PAPS,
of this
[35SLAPS and the remainder of counts were free
Since neither free
[35S]sulfate nor APS was translocated,
these counts must represent products of PAPS breakdown To determine
inside the liposomes.
the orientation of the sulfated proteins,
liposomes were incubated with sulfate-labeled
Analysis
showed that 36% of the counts were associated with
25% were
[35S]sulfate.
acid.
The
reconstituted
[35S]PAPS and the susceptibility
protein to proteases
(acetyltrypsin
1274
of the
and pronase) was
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I/[PAPS](raM}
o
//5
120
I(X)
iiV 80
40 2C 0
I00
2~)0
~0
400
ASO0 I
~'iPAPS] (raM) FIG. i. (A) Double reciprocal plot for the PAPS translocator. The initial rate of translocation in this and in parts b-d was assayed as described under "Materials and Methods." The[35S]PAPS was diluted with buffer to the designated concentration and each assay was done in duplicate. (B) Double reciprocal plot of the effect of 3' ,5'-adenosine dfphosphate on PAPS translocation. The following concentrations of 3',5'-adenoslne diphosphate were used: none (e---e); 0.0025 mM (A---A); 0.01 mM (o---o); and 0.05 (x---x).
examined.
No 35S counts associated with protein fraction in the liposomes
were released by acetyltrypsin liposomes with Triton X-100
treatment,
but prior treatment of the
(0.2%) and subsequent
treatment with proteases
released 46% of the counts into the trichloroacetic indicating
acid-soluble
compartment,
that the sulfated proteins were located inside the reconstituted
liposomes. Substrate by screening
Speclflcity.
The specificity
several nucleotides
of the translocator was examined
(all at 1.0 mM final concentration)
their ability to inhibit translocatlon.
for
We observed more than 80% inhibition
of activity when 3',5'-adenosine
diphosphate was included in the mixture,
both 2',5'-adenosine
and 3',5'-guanosine
25-50% inhibition.
dlphosphate
diphosphate
and
resulted in
Neither ATP, 5'- or 3'-AMP, nor 5'-APS caused significant 1275
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibition of PAPS translocation.
Kinetic analysis showed that both 2',5'-
adenosine diphosphate and 3',5'-guanosine diphosphate were uneompetitive inhibitors and that 3',5'-adenosine diphosphate was the only competitive inhibitor (Fig. IB). At neutral pH PAPS is an anion and since in intact vesicles anion channel inhibitors blocked PAPS transport (27), the effect of these anion transport inhibitors on the reconstituted transporter was investigated. 4,4'-Diisothiocyanostilbene 2,2-disulfonic acid inhibited PAPS translocation 50% at 0.3 mM and 4-acetamide-4'-isothiocyanostilbene produced less inhibition.
2,2'-disulfonic acid
In the reconstituted liposomes, mitochondrial
transport inhibitors, atractyloside, carboxyatractyloside
(16,24), and
palmitoyl-coenzyme A (11,14,25) at physiological concentrations were not inhibitory.
The effects of these inhibitors were different from those
observed by Hirschberg and co-workers for intact Golgi apparatus (2,19), where inhibition was observed at micromolar concentrations.
Moreover, the
affinity of the reconstituted translocstor for PAPS was 1.2 mM as compared to 6 ~
in the intact vesicles.
These differences in the properties may simply
reflect the effects of solubilizing the protein and the fact that the lipid environment within the liposomes is not ideal for the trans]ocator molecule. In summary, the molecule that trsnslocates PAPS has been reconstituted into liposomes.
Purification of this molecule by use of the assay described
should provide definitive information on the properties of the transporter and the role of lipid environment in determining these properties.
ACKNOWLEDGEMENTS We thank Dr. Pamela Talalay for helpful 4iscussion in the preparation of the manuscript and Ms. Diane Emerso~ for her assistance in typing the manuscript.
This study was supported by grants from the Multiple Sclerosis
Society, RG 1411 and RG 1740.
REFERENCES i. 2. 3. 4. 5. 6. 7. 8.
Anholt, R., Lindstrom, J., and Montal, M. (]980) Eur. J. Biochem. 109, 481-487. Capasso, J. M., and Hirschberg, C. B. (1984) J. Biol. Chem. 259, 42634266. DeMarchena, 0., Herndon, R. M., and Guarnieri, M. (1974) Brain Res. 80, 497-502. Farley, J. F., Cryns, D. G., Yang, Y. H. J., and Segel, I.H. (1976) J. Biol. Chem. 251, 4389-4397. Farrell, D. F., and McKhann, G. M. (1971) J. Biol. Chem. 246, 4694-4702. Fleischer, B., and Zambrano, F. (1974) J. Biol. Chem. 249, 5995-6003. Fleischer, B., and Smigel, M. (1978) J. Biol. Chem. 253, 1632-1638. Folch, J., Lees, M., and Sloane, S. G. H. (1957) J. Biol. Chem. 226, 497-509.
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Vol. 155, No. 3, 1988
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Hazelbauer, G. L., and Changeux, J. M. (1974) Proc. Nat. Acad. Sci. U.S.A. 71, 1479-1483. Kilian, P. L., Dunlap, C. R., Mueller, P., Schell, M. A., Huganir, R. L., and Racker, E. (1980) Biochem. Biophys. Res. Commun. 93, 409-414. Lerner, E., Shug, A. L., Elson, C., and Shrago, E. (1972) J. Biol. Chem. 247, 1513-1519. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. Michaelson, D. M., and Raftery, M. A. (1974) Proc. Nat. Acad. Sei. U.S.A. 71, 4768-4772. Pande, S. V., and Blanchaer, M. C. (1971) J. Biol. Chem. 246, 402-411. Reggio, H. A., and Palade G. E. (1978) J. Cell Biol. 77, 288-314. Riccio, P., Aquila, H., and Klingenberg, M. (1975) FEBS Lett. 56, 133138. Rohbins, P. W., and Lipmann, F. (1958) J. Biol. Chem. 233, 681-685. Rosensweig, L. J., and Farquhar, M. G. (1980) Endocrinology 107, 422431. Sehwarz, J. K., Capasso, J. M., and Hirsehberg, C. B. (1984) J. Biol. Chem. 259, 3554-3559. Sugahara, K., and Schwartz, N. B. (1979) Proc. Nat. Acad. Sci. U.S.A. 76, 6615-6618. Sugahara K., and Schwartz, N. B. (1982) Arch. Biochem. Biophys. 214, 589-601. Tennekoon, G., Zaruba, M., and Wolinsky, J. (1983) J. Cell Biol. 97, 1107-1112. Tennekoon, G., Aitchison, S., and Zaruba, M. (1985) Arch. Biochem. Biophys, 240, 932-944. Winkler, H. H., Bygrave, G. L., and Lehninger, A. L. (1968) J. Biol. Chem. 243, 20-28. Woldegiorgis, G., Yousufzai, Y. K., and Shrago, E. (1982) J. Biol. Chem. 257, 14783-14787. Young, R. W. (1973) J. Cell Biol. 57, 175-189. Zoccoli, M. A., and Karnovsky, M. L. (1980) J. Biol. Chem. 255, 11131119.
1277
Vol. 155, No. 3, 1988
Pages 1271-1277
September 30, 1988
RECONSTITUTION OF ADENOSINE 3'-PHOSPHATE 5'-PHOSPHOSULFATE TRANSPORTER FROM RAT BRAIN M.E. Zaruba*, N.B. Schwartz§ and G.I. Tennekoon*
Department of Neurology*, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 and the Department of Pediatrics§, University of Chicago, Chicago, Illinois 60637 Received August 8, 1988
SUMMARY: Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the "active" sulfate donor for sulfated macromolecules, is synthesized in the cytosolle fraction of rat brains. This molecule is then translocated into the lumen of the Golgl apparatus so that it is available to the sulfotransferase enzymes. The protein responsible for the PAPS translocatlng activity has been solubillzed from vesicles enrlehed in enzyme markers for the Golgi apparatus and reconstituted into llposomes. In reconstituted liposomes translocatlng activity has a pH optimum of 7.0 and activity was increased 3-fold by divalent cations, although EDTA produced no inhibition. The affinity of the reconstituted translocator for PAPS showed a Km of 1.2 m M w i t h a Vmax of 14 pmol of PAPS translocated/mln/mg of protein. Specificity of the translocator activity was tested with a number of nucleotlde analogues and only 3',5'adenosine diphosphate was a competitive inhibitor. Inhlbitors of the mltochondrial ADP/ATP transporter and the red cell anion channel blocked transport of PAPS only at very high concentrations. © 19s8AcaaemioPress, ~nc.
Sulfation of macromolecules takes place largely in the Golgi complex (6,7,15,18,26).
For example, cerebroside sulfotransferase, the enzyme that
catalyzes the sulfation of galactocerebroside, was found to be localized to the Golgi apparatus in brain and kidney (5-7) and had a luminal orientation in trans-Golgl (22). The enzymes involved in the synthesis of the active sulfate donor adenosine 3'-phosphate 5'-phosphosulfate sulfurylase and adenosine 5'-phosphosulfate
(PAPS), ATP-
(APS)kinase, had been localized
to the cytosol in yeast (17) and Hirschberg and co-workers (2,19) have shown the presence of a translocatlng molecule for PAPS in intact Golgi vesicles from liver. For an understanding of the mechanism of regulation of sulfatlon in rat brain, it will be necessary to purify the PAPS transporter.
Abbreviations used:
A prerequisite
APS, adenosine 5'-phosphosulfate; PAPS, adenosine 3'-
phosphate 5'-phosphosulfate.
0006-291)<)88 $1.50 1271
Copyright © 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
for purifying this "carrier" molecule is the ability to solubilize and reconstitute them in liposomes.
In this communication, we describe the
reconstitntion into liposomes of the PAPS translocating molecule and the subcellular localization of the PAPS synthesizing enzymes in rat brain.
MATERIALS AND METHODS Sodium cholate, sodium phosphate, soybean lipids, mercuric chloride, ammonium acetate, acetyltrypsin and soybean trypsin inhibitor, 4,4'diisothiocyanostilbene 2,2'-disulfonic acid and palmitoyl-CoA were obtained from Sigma. Triton X-100 was purchased from Rohm and Haas. All the nucleotides used in this study were purchased from P.L. Biochemicals. [3H]Methoxy inulin (5,000 Ci/mmol) was purchased from ICN Radiochemicals. [35S]Adenosine 3'-phosphate 5'-phosphosulfate (1.2-2.8 mCi/mmol), [35S]sodium sulfate and [32p]Na4P207 (I-60 Ci/mmol) were obtained from New England Nuclear. Microsome Vesicle Preparation. The microsome vesicles (Golgi-enriched) were prepared as described (22), from brains of i0- to 20-day-old SpragueDawley rats (Charles River, MA). Reconstitution of Vesicles. The reconstitution of microsomal protein into liposome vesicles was based on procedures previously described (1,9,10,13). Microsomal proteins (3.0 mg/ml) were solubilized for 1 h at 4°C in I0 mM sodium phosphate, I00 mM NaCI, pH 7.4 (PBS) containing 2% sodium cholate, and 0.2% crude soybean lipids. After solubilization, the protein was diluted to 1.3 mg/ml with 2% sodium cholate containing 2% soybean lipids, sonicated for 30 sec, and then dialyzed against PBS containing 10 mM sodium azide (four changes each of 50 volumes over 60 h). Transporter Assay. Transport activity was measured by adding l0 ~l of 0.2 mM [35S]PAPS (400,000) dpm) to 200 ~I of reconstituted liposomes (I-2 mg/ml of protein) to which i0 ~l of 200 mM CaCI 2 had been added (final concentration of CaCI 2, 10 mM), and incubation at 20°C or as indicated. The assay was stopped at specified times by adding I0 ~l of 200 mM HgC12 and placing the mixture on ice. The incubation mixture was then added to a 2.0ml Sephadex G-100 column equilibrated and then eluted with PBS. The 5-drop column fractions were collected and counted in 5.0 ml of liquiscint (National Diagnostics). This procedure provides good separation of the liposomeassociated [35S]PAPS from the "free" [35S]PAPS. Identification of 35S-labeled Materials. Twelve 200-~1 aliquots of reconstituted vesicles were incubated as described for the transporter assay. The vesicle fractions, separated from the "free" PAPS on the Sephadex G-100 columns, were pooled, frozen and lyophilized. The lyophillzed sample was brought to 1.0 ml with ethanol:H20, l:l (v:v) and 20-~1 aliquots were spotted (23) in duplicate on Whatman No. 3 paper with appropriate nucleotide markers and developed (ascending) for 4 h in I M ammonium acetate:ethanol (7:3, v/v), pH 7.5. The products were then visualized by UV light, cut out, extracted for 30 min with l.O ml of H20, and counted in lO ml of liquiscint (National Diagnostics). For identification of 35SO4-1abeled lipid and proteins, a 0.5-ml aliquot of the lyophilized vesicle sample in ethanol:H20, I:I (v/v) was extracted in 5 ml of chloroform:methanol, i:i (v/v) for 60 min at 4°C and then centrifuged at 400 x g for 25 mln at 4°C. The supernatant fraction was decanted and saved. The pellet was again extracted and centrifuged and the supernatant fractions were pooled, subjected to lipid extraction, and partitioned according to Folch et al. (8). The pellet remaining from the chloroform:methanol extraction was washed three times with I0 ml of 6% trichloracetic acid and 0.5% phosphotungstic acid, with centrifugation for 15 min at 4°C at 300 x g between washes. It was then solubillzed in 1.0 ml of N,C.S. tissue solubilizer (Amersham), 1272
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
decolorized with I00 ~i of 30% hydrogen peroxide overnight at 42°C, and counted by liquid scintillation counting. Sensitivity of Reconstituted Transporter to Acetyltrypsin. For each time point, 200 BI of reconstituted vesicles (0.26 mg of protein) were preincubated for specified times at 20°C with i0 ~I of acetyltrypsln (i mg/ml). Then I0 ~I of PBS containing 5 mg/ml of soybean trypsin inhibitor were added, the sample was mixed with a Vortex mixer and assayed for PAPS transporter activity by the transporter assay described above. Controls with buffer instead of acetyltrypsln were included. Enzyme Assays. Subcellular fractionation was performed as previously established in our laboratory (3). Standard definitions are used for mitochondrial, microsomal and cytosolic fractions. ATP sulfurylase was assayed by measuring the formation of [32p]ATP from adenosine 5-phosphosulfate and 32p-labeled pyrophosphate as described by Sugahara and Schwartz (4,20,21). APS kinase was assayed by measuring conversion of [35S]APS to [35S]PAPS as described by Sugahara and Schwartz (20). Protein Determination. Proteins were assayed by the method of Lowry et al. (12) with bovine serum albumin as standard.
RESULTS AND DISCUSSION Localization of Enzymes that Synthesize Adenosine 3'-Phosphate 5'Phosphosulfate.
The specific activities of ATP-sulfurylase and APS-kinase
were determined in homogenates and various subcellular fractions (Table I). Specific activities were highest in the supernatant fractions, establishing that the enzymes responsible for the synthesis of PAPS were localized in the cytosol of cells, similar to those reported for yeast.
Hence, PAPS must be
translocated into the Golgi complex so that it is available to the sulfotransferase. Reconstitution of PAPS Translocator.
We first ensured that the PAPS
translocator was present in Golgi-enriched microsomes
(data not shown).
We
next tested a variety of detergents and found that rat brain microsomes were extracted only with 2% sodium cholate or 1.5% octylglucoside.
Both
detergents extracted 60% of the total microsomal proteins, which showed identical PAPS translocating activities,
and in all subsequent experiments
the initial extraction was carried out with 2% sodium cholate and proteins were reconstituted with 0.2% soybean lipids as described under "Materials and Methods." Characterization of PAPS Translocating Activity in Reconstituted Liposomes.
Both [2-3H]methoxyinulin
and carrier-free
[35S]sulfate were
excluded from the liposomes, suggesting that the liposomes were not leaky to small molecular weight compounds.
Moreover, no translocation of [35S]PAPS
was observed in the absence of extracted protein fraction, or in the presence of other protein fractions, including bovine serum albumin, protein extracts from red cell ghosts, or proteins obtained by extraction of boiled microsomes.
The translocating activity was decreased by about 70% by
1273
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TABLE 1.
Subcellular localization of PAPS synthesizing enzymes in rat brain
Fraction
Specific Activity APS Kinase ATP Sulfurylase cpm/mg protein/min 104
Homogenate 1000 x g supernatant i000 x g pellet (nuclei) 12,000 x g supernatant 12,000 pellet x g (mitochondria) 00,000 x g supernatant (cytosol) i00,000 x g pellet (microsomes)
1.51 1.95 1.40 4.96 1.37 8.96 0.86
1.50 1.50 0.45 3.93 0.67 5.72 0.54
Whole brains from 14-day-old Sprague-Dawley rats were removed and homogenized by use of a Dounce homogenizer. The homogenate was centrifuged at 1,000 x g for i0 min, the pellet (nuclei) was washed once, and the supernatant fractions were combined and centrifuged at 12,000 x g for 30 min. The pellet (mitochondria) was washed and the supernatant franctions were combined and centrifuged at i00,000 x g for 90 min. The supernatant fractions were designated as cytosol and the pellet constituted the microsomal fraction. Details of the assays are described in the text.
treatment with acetyltrypsin,
suggesting
that the translocating molecule is a
protein. In the reconstituted
liposomes,
the rate of translocation
of PAPS was
linear for the first 7 min, after which the rate decreased but still remained significant.
Translocation
concentration about 7.0.
activity was proportional
(8-154 Bg), temperature-dependent,
There was demonstrable
added divalent
cations,
slight stimulatory
translocating
to protein
and showed a pH optimum of activity in the absence of
and EDTA did not inhibit translocation,
effect.
Nevertheless,
but had a
divalent cations increased
the rate
of PAPS translocation by 2- to 3-fold, with calcium having the greatest stimulatory
effect.
concentrations
Transport of [35S]PAPS as a function of different
of this compound showed typical saturation kinetics and a
double reciprocal plot gave a linear relationship with a Km=l.2 mM and a Vmax=14 pmol of PAPS translocated/min/mg reconstituted
transporter
Identification counts translocated
is different
of protein
(Fig.
IA).
The K m of the
from that in intact vesicles
of 35S-labeled Material.
(6 BM).
Analysis of the [35S]PAPS
showed that about half the counts were associated with
protein and very few (about 1%) of the counts with the lipid fraction. remaining
counts were soluble in 10% trichloroacetic
soluble material [35S]PAPS,
of this
[35SLAPS and the remainder of counts were free
Since neither free
[35S]sulfate nor APS was translocated,
these counts must represent products of PAPS breakdown To determine
inside the liposomes.
the orientation of the sulfated proteins,
liposomes were incubated with sulfate-labeled
Analysis
showed that 36% of the counts were associated with
25% were
[35S]sulfate.
acid.
The
reconstituted
[35S]PAPS and the susceptibility
protein to proteases
(acetyltrypsin
1274
of the
and pronase) was
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I/[PAPS](raM}
o
//5
120
I(X)
iiV 80
40 2C 0
I00
2~)0
~0
400
ASO0 I
~'iPAPS] (raM) FIG. i. (A) Double reciprocal plot for the PAPS translocator. The initial rate of translocation in this and in parts b-d was assayed as described under "Materials and Methods." The[35S]PAPS was diluted with buffer to the designated concentration and each assay was done in duplicate. (B) Double reciprocal plot of the effect of 3' ,5'-adenosine dfphosphate on PAPS translocation. The following concentrations of 3',5'-adenoslne diphosphate were used: none (e---e); 0.0025 mM (A---A); 0.01 mM (o---o); and 0.05 (x---x).
examined.
No 35S counts associated with protein fraction in the liposomes
were released by acetyltrypsin liposomes with Triton X-100
treatment,
but prior treatment of the
(0.2%) and subsequent
treatment with proteases
released 46% of the counts into the trichloroacetic indicating
acid-soluble
compartment,
that the sulfated proteins were located inside the reconstituted
liposomes. Substrate by screening
Speclflcity.
The specificity
several nucleotides
of the translocator was examined
(all at 1.0 mM final concentration)
their ability to inhibit translocatlon.
for
We observed more than 80% inhibition
of activity when 3',5'-adenosine
diphosphate was included in the mixture,
both 2',5'-adenosine
and 3',5'-guanosine
25-50% inhibition.
dlphosphate
diphosphate
and
resulted in
Neither ATP, 5'- or 3'-AMP, nor 5'-APS caused significant 1275
Vol. 155, No. 3, 1988
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
inhibition of PAPS translocation.
Kinetic analysis showed that both 2',5'-
adenosine diphosphate and 3',5'-guanosine diphosphate were uneompetitive inhibitors and that 3',5'-adenosine diphosphate was the only competitive inhibitor (Fig. IB). At neutral pH PAPS is an anion and since in intact vesicles anion channel inhibitors blocked PAPS transport (27), the effect of these anion transport inhibitors on the reconstituted transporter was investigated. 4,4'-Diisothiocyanostilbene 2,2-disulfonic acid inhibited PAPS translocation 50% at 0.3 mM and 4-acetamide-4'-isothiocyanostilbene produced less inhibition.
2,2'-disulfonic acid
In the reconstituted liposomes, mitochondrial
transport inhibitors, atractyloside, carboxyatractyloside
(16,24), and
palmitoyl-coenzyme A (11,14,25) at physiological concentrations were not inhibitory.
The effects of these inhibitors were different from those
observed by Hirschberg and co-workers for intact Golgi apparatus (2,19), where inhibition was observed at micromolar concentrations.
Moreover, the
affinity of the reconstituted translocstor for PAPS was 1.2 mM as compared to 6 ~
in the intact vesicles.
These differences in the properties may simply
reflect the effects of solubilizing the protein and the fact that the lipid environment within the liposomes is not ideal for the trans]ocator molecule. In summary, the molecule that trsnslocates PAPS has been reconstituted into liposomes.
Purification of this molecule by use of the assay described
should provide definitive information on the properties of the transporter and the role of lipid environment in determining these properties.
ACKNOWLEDGEMENTS We thank Dr. Pamela Talalay for helpful 4iscussion in the preparation of the manuscript and Ms. Diane Emerso~ for her assistance in typing the manuscript.
This study was supported by grants from the Multiple Sclerosis
Society, RG 1411 and RG 1740.
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