From the
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.
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
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.
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
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
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.
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).
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
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
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
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.
Molecular sizes were calculated from Figs. 1, 2, 3, and 5. In all
cases, n = 3.
We thank Karen Welch and Annette Stratton for
excellent secretarial assistance.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)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.
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.
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.
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.
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.
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.
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).
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).
(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.
Table: Molecular sizes of S. cerevisiae Golgi GDPase
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.