©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Yeast Golgi Glycosylation
GUANOSINE DIPHOSPHATASE FUNCTIONS AS A HOMODIMER IN THE MEMBRANE (*)

Patricia Berninsone (1), Zhen-Yuan Lin (1), Ellis Kempner (2), Carlos B. Hirschberg (1)(§)

From the (1)Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, Massachusetts 01655-1013 and (2)Laboratory of Physical Biology, NIAMS, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Golgi lumenal GDPase plays an important role in the mannosylation of proteins and lipids of Saccharomyces cerevisiae by regulating the amount of GDP-mannose available in the Golgi lumen. The enzyme makes available GMP as an antiporter to be coupled with entry of GDP-mannose into the Golgi lumen from the cytosol. Using radiation inactivation and target analysis, we have now determined the functional molecular mass of the GDPase within the Golgi membrane and whether or not the enzyme has functional associations with other Golgi membrane proteins, including mannosyltransferases and the GDP-mannose transporter. The functional size of the GDPase was found to be approximately twice the estimated structural target size of the protein; this strongly suggests that the GDPase protein in situ functions as homodimer and does not require association with other membrane proteins for its function.


INTRODUCTION

The addition of mannose to N- and O-linked outer chain oligosaccharides of glycoproteins and mannosylation of glycosphingolipids occurs in the yeast Golgi apparatus lumen(1) . These reactions require a mannosyl donor, GDP-mannose, which is synthesized in the cytosol and must be translocated to the Golgi lumen by a specific membrane carrier(2) . Following mannosylation of proteins and lipids, the other reaction product, GDP, is converted to GMP by a specific lumenal GDPase. By analogy with the mammalian transport system, this is the putative antiporter for GDP-mannose entry in yeast(2) . Previous studies in vivo and in vitro have shown that the GDPase plays a pivotal role in the regulation of Golgi lumenal mannosylation reactions in Saccharomyces cerevisiae. The enzyme has previously been purified and characterized, and its gene, GDA-1 has been cloned(3, 4) . The purified deglycosylated GDPase migrates on SDS-PAGE()with an apparent molecular weight of 47,000(3) . The deduced protein sequence of the GDA-1 gene encodes a 519-amino acid polypeptide with a calculated mass of 57 kDa containing three potential N-glycosylation sites(4) . It is a typical type II membrane protein with a single hydrophobic domain, which acts as an uncleaved signal sequence and membrane anchor, and is preceded by a short hydrophilic cytosolic tail.

In vivo, null mutants of the GDA-1 gene, gda1, show a major reduction in glycosylation of Golgi O- and N-mannosylated proteins as well as mannosylated inositolphosphoryl ceramides(4) . Studies in vitro with Golgi vesicles derived from wild-type and null mutants have shown the rate of entry of GDP-mannose into vesicles is significantly reduced in null mutants, as predicted(5) . This strongly suggests that the impaired mannosylation in vivo in null mutants is due to a decrease in GMP, the putative antiporter, and consequent reduced availability of GDP-mannose in the Golgi lumen(5) .

Given the above described novel physiological relevance of the Golgi lumenal GDPase in glycosylation reactions, an important question is whether or not the GDPase has functional associations with other Golgi proteins (i.e. mannosyltransferases and GDP-mannose transporter) and how the GDPase functions in the membrane. One of the most powerful approaches to characterize functional protein-protein interactions in situ is radiation inactivation and target size analyses of proteins(6) . This approach permits determination of the molecular mass of the functional unit being studied and can provide evidence of multimeric functional association(s) of a given protein within its environment(7) .

In this study, using radiation inactivation of wild-type and null mutant membranes, the functional size of GDPase is approximately twice the estimated structural target size of the GDA-1 protein. This strongly suggests that in situ the GDPase functions as a homodimer and its function does not require association with other membrane proteins such as mannosyltransferases or the GDP-mannose transporter.


MATERIALS AND METHODS

Strains and Preparation of Membrane Fractions

S. cerevisiae strains G2-25 (ura 3-52, lys 2-801 am, ade2-101 oc, trp1-101, his3-200, leu2-1) and G2-28 (ura 3-52, lys 2-801 am, ade2-101 oc, trp1-101, his3-200, leu2-1, gda1::LEU2) (4) were used as wild-type and null-mutant for GDA-1, respectively.

For each irradiation series, membranes were prepared from 4 liters of cells grown in YEPD medium at 30 °C to an OD of 4. Cells were collected by centrifugation, washed with aqueous 10 mM azide, suspended in cryoprotective buffer (8) (1 ml/g, wet weight), to which glass beads were added, and broken by four 30`` bursts with a Wizard vortex at 5,000 rpm. The homogenate was centrifuged for 5 min at 2,600 g; the pellet was resuspended in cryobuffer, and the cell breakage and centrifugation were repeated once. The low speed supernatants were combined and centrifuged for 40 min at 100,000 g. The pellet (membrane fraction) was resuspended in cryobuffer at a protein concentration of 7-10 mg/ml. After adding 400 units/ml glucose-6-phosphate dehydrogenase (from Leuconostoc mesenteroides, Sigma), 500-µl portions of the resulting suspension were dispensed into 2-ml glass ampules and immediately frozen on dry ice. The ampules were sealed with an air/butane torch and stored at -80 °C until shipped for irradiation.

Irradiation/Manipulation of Samples after Irradiation

Irradiation was performed as previously described(9) . Following irradiation, the samples were returned on dry ice. Upon receipt, each ampule was opened; the overlying gas was displaced with N, and the samples were allowed to thaw in an ice bath. GDPase activity in S. cerevisiae membranes was found to be stable to several cycles of freezing and thawing; however, to minimize the possibility of freeze-thaw artifacts, all thawed samples were separated into aliquots, which were immediately refrozen and stored at -80 °C until they were used.

Enzyme Assays

GDPase activity was assayed as previously described(3) . Briefly, incubation mixtures contained enzyme (3-100 µg of membrane extract protein), CaCl (1 µmol), Triton X-100 (100 µg), GDP (0.2 µmol), and Imidazole-HCl buffer, pH 7.6 (20 µmol). To measure ADP hydrolysis, GDP was replaced by ADP at the same concentration. Glucose-6-phosphate dehydrogenase was assayed by recording the rate of NADPH formation at 340 nm at 25 °C and pH 7(10) .

Preparation of GDPase Antiserum

Polyclonal rabbit antibodies were raised against a fusion protein of the N-terminal domain (27 kDa) of glutathione S-transferase, fused with the C-terminal domain of S. cerevisiae GDPase (53 kDa). The latter includes amino acid residues 46-518, excluding only the transmembrane domain and the 9-amino acid cytosolic tail. The glutathione S-transferase-GDPase fusion expression plasmid (p46-5) was constructed so that the GDPase sequence was in frame with glutathione S-transferase sequence. Briefly, a 1.4-kilobase fragment was generated by polymerase chain reaction from p13H (4) using the following primers: primer A, nucleotide sequence coding from Lys-46 to Pro-55 with a 5`-overhang containing a BamHI site and a C/G clamp; primer B, nucleotides complementary to sequence coding from Asp-509 to Ala-518, with a 5`-overhang containing a stop anticodon, EcoRI site, and C/G clamp.

The resulting polymerase chain reaction product was purified, digested with BamHI and EcoRI, and cloned into the BamHI/EcoRI sites of pGEX-2 (Promega). DH5 Escherichia coli was transformed with p46-5 according to Maniatis et al.(11) . Transformants induced with isopropyl-1-thio--D-galactopyranoside overexpressed an insoluble 80-kDa protein, which was solubilized in 7.3 M urea from the inclusion bodies after breaking of cells with a French press. Following renaturation by dialysis, the fusion protein was purified by affinity chromatography on glutathione-Sepharose(12) .

Immunodetection of GDPase Protein

GDPase protein was denatured with 0.5% SDS, 1% 2-mercaptoethanol, and 0.1% Triton X-100 at 100 °C for 3 min. Endoglycosidase H (a generous gift from Dr. R. Trimble, NY State Department of Health) was added (5 units/µg protein), and samples were digested in 50 mM citrate buffer, pH 5.5, containing protease inhibitor for 8 h at 37 °C.

Samples (untreated or endoglycosidase H digested) were fractionated by SDS-PAGE(13) , and proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Millipore). These were blocked in TBS (20 mM Tris-HCl, pH 7.5, 0.5 M NaCl) containing 3% gelatin and 1% milk. After rinsing twice with TTBS (TBS containing 0.05% Tween 20), membranes were incubated in a 1:5,000 dilution of a-GDPase antiserum in TTBS, 1% milk, 1% gelatin for 2 h, rinsed with TTBS, and incubated in a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Promega). Detection was performed using the ECL system (Amersham Corp.). For each membrane, several exposures were made on Reflections film (DuPont NEN).

Relative amounts of GDPase protein were calculated based on the relative intensities of the bands on the films(14, 15) . Standard curves were run using dilutions of non-irradiated membrane extracts. The films were scanned using a Soft Laser Scanning Densitometer (Biomed Instruments, Inc.) with a 3-mm slit, and for each band the integrated area was determined with the accompanying software.


RESULTS

Functional Target Size of the GDPase Activity

For simple target analysis to be valid in these experiments it was important to show that the affinity of the enzyme for GDP was not altered by radiation exposure. The K of the GDPase was determined in membranes that had been exposed to either 0 or 36 megarads. The values were virtually identical (1.5 versus 1.0 µM), demonstrating that no GDPases with altered activity were generated by irradiation.

Knowledge of the functional target size of the GDPase activity is important to determine whether the protein functions as a monomer or oligomer in the membrane and whether association with other proteins is necessary for its function in situ. Following irradiation of membranes from wild type and gda1 null mutants, two different approaches were used to determine the functional target size of the GDPase activity. In one case, GDPase activity was calculated as the difference in hydrolysis of GDP for each irradiation dose between membranes from wild-type and the null mutants. As can be seen in Fig. 1, the activity decreased exponentially with radiation dose. The calculated target size for the GDPase was 120 ± 17 kDa (). As will be shown below, the higher standard deviation for the target size obtained with this approach was expected as it involved the difference in activity determinations of two completely independent sets of irradiated samples. In all these experiments, glucose-6-phosphate dehydrogenase was added as an internal standard(16, 17) . The calculated target size for this enzyme was 126 ± 18 kDa (n = 8), while the true size is 106 kDa; because of this difference, the target sizes for the GDPase were also calculated following correction by the ratio between the true and observed target sizes of the glucose-6-phosphate dehydrogenase in each experiment. This standardized target size for the GDPase, measured as the difference in GDP hydrolysis between wild-type and null mutant, was 103 ± 16 kDa (n = 3).


Figure 1: GDPase activity after exposure of membranes of S. cerevisiae to radiation. Membranes from wild-type and null mutants were prepared, irradiated, and assayed for GDPase activity as described ``Materials and Methods.'' Remaining GDPase activity was determined for each irradiation dose as the difference in GDP hydrolysis between membranes for wild-type and null mutant cells. Symbols represent different independent irradiation experiments.



Alternatively, GDPase activity was measured in wild-type membranes as the difference between hydrolysis of GDP and ADP, the latter a substrate for nonspecific phosphatases. These measurements also resulted in an exponential loss of net GDPase activity with radiation dose, as seen in Fig. 2, yielding a target size of 132 ± 6 kDa (). The GDPase target size calculated as the difference between GDP and ADP corrected for the glucose-6-phosphate dehydrogenase target size was 106 ± 11 kDa (n = 3). In irradiated null mutant membranes, no significant differences were found between hydrolysis of GDP and ADP (data not shown).


Figure 2: GDPase activity after exposure of membranes of S. cerevisiae to radiation. Membranes from wild-type cells were prepared, irradiated, and assayed as described under ``Materials and Methods.'' Remaining GDPase activity was determined for each irradiation dose as the difference between hydrolysis of GDP and ADP in wild-type membranes. Symbols represent different independent irradiation experiments.



Using two completely independent means of subtracting nonspecific GDP hydrolysis, no significant differences were observed in the GDPase inactivation curves and resulting target sizes. These results demonstrate that no other GDPase activity exists in these membranes.

Target Size of the Structural Unit of GDPase

To determine the target size of the structural unit of the GDPase, a polyclonal rabbit antiserum was raised against a glutathione S-transferase-GDPase fusion protein expressed in E. coli. As shown in Fig. 3, the antiserum was highly specific and reacted with a 55,000 M protein in wild-type membranes treated with endoglycosidase H prior to SDS-PAGE and Western blots. This band was not detected in membranes from a GDA-1 null mutant strain, which had been subjected to the same experimental protocol, demonstrating the high degree of specificity of the antiserum. The lower molecular weight bands detected are observed both in wild-type and null mutant membranes; this strongly suggests that they are cross-reacting proteins and not proteolysis products of the GDPase.


Figure 3: Immunodetection of GDPase protein in S. cerevisiae membranes. Untreated (-) or endoglycosidase H treated (+) proteins from membranes of S. cerevisiae were subjected to Western blot analysis as described under ``Materials and Methods.'' WT, wild type; (gda1), GDA-1 null mutant.



In previous studies, we showed that the purified deglycosylated GDPase had an apparent molecular weight on SDS-PAGE of 47,000(3) , while the molecular weight of the predicted polypeptide from the open reading frame was 57 kDa(4) . As shown in Fig. 3, the size of the immunoreactive band reported here is in close agreement with the predicted size from the open reading frame and strongly suggests that the previously reported 47,000 M protein may have been the result of proteolytic cleavage of the enzyme during purification. The polypeptide of the GDPase predicted from the open reading frame of the GDA-1 gene contains three potential glycosylation sites. Previously, we had shown that the enzyme binds to a concanavalin A-Sepharose column yet behaved differently on a Mono Q column after deglycosylation(3) . Untreated, wild-type membranes showed a broader area of immunoreactivity with slower mobility on Western blots under the condition described here (Fig. 3), probably representing the GDPase protein with different degrees of glycosylation.

The loss of immunoreactive GDPase protein as a function of radiation was determined by performing Western blot analyses of endoglycosidase H-treated wild-type irradiated membranes. A representative Western blot is shown in Fig. 4. To ensure that the amount of protein measured was proportional to the actual quantity present, a set of calibration curves was run. Under the conditions described here, the immunointensity of the 55,000 M band was linear over a 10-fold concentration range of protein. Following irradiation of samples, simple exponential decrease in the intensity of immunoreactivity labeling of this band can be observed ( Fig. 4and 5), from which a target size of 76 ± 13 kDa (n = 3) was calculated. When this target size was further corrected for the ratios of the observed and true target sizes of the glucose-6-phosphate dehydrogenase, a target size of 65 ± 9 kDa was obtained.


Figure 4: Immunoreactive GDPase protein from irradiated S. cerevisiae membranes. Proteins from irradiated wild-type membranes were digested with endoglycosidase H and subjected to Western blot analysis of the GDPase protein as described under ``Materials and Methods.'' A representative experiment is shown. Doses are shown in megarads (Mrad).




DISCUSSION

In this study, we have determined the functional as well as structural target sizes of the in situ Golgi GDPase of S. cerevisiae. Using two different estimations of nonspecific hydrolysis of GDP, that was observed in the mutant as well as with ADP as nonspecific substrate, the functional target size of the GDPase was approximately 120 kDa (). Western blots with a highly specific antiserum were used to determine the structural unit size of the enzyme as approximately 60 kDa (). This value is in close agreement with the previously observed open reading frame of the protein () and the calculated size of the immunoreactive deglycosylated protein ().

Target size determinations by radiation inactivation assume complete inactivation of irradiated molecules, while undamaged ones remain completely active. Therefore, irradiated enzymes show a decrease in V without change of K(6). The simple inactivation curves, as reported here for the functional target size of the GDPase, show that a single size population of molecules is responsible for the GDPase activity in the membrane and that no other large proteins participate in either activating or inhibiting the activity in situ (see below).

Two models may account for a functional target size that is twice the structural target size(7) . In both, the protomer is associated with another polypeptide within the membrane; the other polypeptide is either identical to the protomer or different but has a very similar molecular mass. Each one of the two peptides is required for function, and radiation damage to one results in loss of activity of both. We believe that strong evidence supports the homodimer model to occur in situ. In a previous study, following native electrophoresis and active staining of the GDPase, a protein from that region was recovered and subjected to SDS-PAGE, where it migrated as 47,000 M(3) . We now know that this protein is a proteolytic cleavage product of the 55-kDa protein; if the native GDPase was a heterodimer, one would expect two proteins of different size unless one of them has an M of 47,000 in the native state. In this instance, however, one would expect peptide sequencing and cloning to have shown two separate proteins. All peptides that were sequenced were found in the open reading frame of the cloned protein(4) , ruling out a heterodimer.

The dimeric target size of the GDPase strongly suggests that the enzyme in the Golgi membrane is not coupled, in a functional manner, to mannosyl transferases or the GDP-mannose transporter (proteins that provide substrates or products for the GDPase (2) or to any other protein of unknown function. However, the fact that a functional coupling does not occur does not exclude that all of these proteins may be part of complexes within the Golgi membrane.

Radiation inactivation has been previously used to determine the functional size of several mammalian Golgi proteins. The majority have yielded functional target sizes twice the size of the respective structural units, consistent with them functioning in situ as homodimers; examples are the UDPase(18) , which appears to have a similar functional role in the Golgi membrane of mammals as the yeast GDPase, and galactosyl- and sialyltransferases(18) , as well as the recently purified adenosine 3`-phosphate 5`-phosphosulfate transporter (19). Other mammalian Golgi proteins such as the heparan sulfate N-deacetylase/N-sulfotransferase have been shown to function as a monomer(20) . Thus, there appears to be no functional oligomeric pattern of proteins in the Golgi membrane. The Golgi apparatus of S. cerevisiae is a complex organelle, and current models point to a highly organized biochemical and functional subcompartmentation. A growing number of specific marker proteins for this organelle are being identified and their genes cloned. Target analysis of these proteins will allow a better understanding of their functional organization within the S. cerevisiae Golgi membrane.

  
Table: Molecular sizes of S. cerevisiae Golgi GDPase

Molecular sizes were calculated from Figs. 1, 2, 3, and 5. In all cases, n = 3.



FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM 30365. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, 55 Lake Ave. North, Worcester, MA 01655. Tel.: 508-856-2450; Fax: 508-856-6231.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; TBS, Tris-buffered saline.


ACKNOWLEDGEMENTS

We thank Karen Welch and Annette Stratton for excellent secretarial assistance.


REFERENCES
  1. Herscovics, A., and Orleans, P.(1993) FASEB J.7, 540-550 [Abstract/Free Full Text]
  2. Abeijon, C., Orlean, P., Robbins, P. W., and Hirschberg, C. B.(1989) Proc. Natl. Acad. Sci. U. S. A.86, 6935-6939 [Abstract]
  3. Yanagisawa, K., Resnick, D., Abeijon, C., Robbins, P. W., and Hirschberg, C. B.(1990) J. Biol. Chem.265, 19351-19355 [Abstract/Free Full Text]
  4. Abeijon, C., Yanagisawa, K., Mandon, E., Hausler, A., Moremen, K., Hirschberg, C. B., and Robbins, P. W.(1993) J. Cell Biol.122, 307-323 [Abstract]
  5. Berninsone, P., Miret, J. J., and Hirschberg, C. B.(1994) J. Biol. Chem.269, 207-211 [Abstract/Free Full Text]
  6. Kempner, E. S.(1993) Trends Biochem. Sci.18, 236-239 [Medline] [Order article via Infotrieve]
  7. Kempner, E. S., and Fleischer, S.(1989) Methods Enzymol.172, 410-439 [Medline] [Order article via Infotrieve]
  8. Beliveau, R., Demeule, M., Ibnoul-Khatib, H., Bergeron, M., Beauregard, G., and Potier, M.(1988) Biochem. J.252, 807-813 [Medline] [Order article via Infotrieve]
  9. Harmon, J. T., Nielsen, T. B., and Kempner, E. S.(1985) Methods Enzymol.177, 65-94
  10. Kempner, E. S.(1988) Adv. Enzymol.61, 107-147 [Medline] [Order article via Infotrieve]
  11. Maniatis, T., Fritsch, E. F., and Sambrook, J.(1989) in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  12. Smith, D. B., and Johnson, K. S.(1988) Gene (Amst.) 67, 31-40 [CrossRef][Medline] [Order article via Infotrieve]
  13. Laemmli, U. K.(1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  14. Stevens, B. R., Fernandez, A., Hirayama, B., Wright, E. M., and Kempner, E. S.(1990) Proc. Natl. Acad. Sci. U. S. A.87, 1456-1460 [Abstract]
  15. Straka, J. G., Bloomer, J. R., and Kempner, E. S.(1991) J. Biol. Chem.266, 24637-24641 [Abstract/Free Full Text]
  16. Lai, F. A., Lo, M. M. S., and Barnard, E. A.(1987) in Target Size Analysis of Membrane Proteins (Venter, J. C., and Jung, C. Y., eds), pp. 33-41, A. R. Liss New York
  17. Norby, J. G., and Jensen, J.(1989) J. Biol. Chem.264, 19548-19568 [Abstract/Free Full Text]
  18. Fleischer, B., McIntyre, J. O., and Kempner, E. S.(1993) Biochemistry32, 2076-2081 [Medline] [Order article via Infotrieve]
  19. Mandon, E. C., Milla, M., Kempner, E. S., and Hirschberg, C. B.(1994) Proc. Natl. Acad. Sci. U. S. A.91, 10707-10711 [Abstract/Free Full Text]
  20. Mandon, E. C., Kempner, E. S., Ishihara, M., and Hirschberg, C. B. (1994) J. Biol. Chem.269, 11729-11733 [Abstract/Free Full Text]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.