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THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
0
1985
by
The
American
Society
of Biological
Chemists,
Inc.
Vol.
260,
No.
11,
Issue
of
June
10,
pp.
6600-6608
1985
Printed
in
Lj.S.A.
The Transport
and
Utilization
of
Acetyl Coenzyme
A
by Rat Liver
Golgi Vesicles
0-ACETYLATED SIALIC ACIDS ARE
A
MAJOR PRODUCT*
(Received for publication, December
3,
1984)
Ajit VarkiS and Sandra Diaz
From
the Department
of
Medicine, UCSD Cancer Center, University
of
California at Sun Diego, Sun Diego, California
92093
When intact
rat
liver Golgi vesicles were incubated
with [~cetyZ-~H]acetyl coenzyme
A,
radioactivity was
incorporated into the vesicles in a manner dependent
upon temperature, time, protein, and acetyl-coA con-
centration. The vesicles concentrated the label 121-
fold relative to the medium within 20 min, suggesting
an active transport mechanism operating in intact ves-
icles, and incorporated more than
50%
of this label into
acid-insoluble materials. This was supported by the
finding that incorporation was markedly reduced by
Triton
X-100
at
levels above
its
critical micellar con-
centration. While the intravesicular low molecular
weight fraction was predominantly free acetate, ace-
tate ions themselves were not permeant to the vesicles.
Double-label experiments suggested that the transport
process involved the entire acetyl-coA molecule. This
was further supported by the fact that coenzyme ASH,
palmitoyl-CoA and butyryl-CoA were markedly inhib-
itory. Incorporation was optimal at 22
OC
at
pH
7.0,
and was moderately stimulated by ATP. However,
compounds known to abolish proton gradients or to
inhibit the Golgi proton pump had no effect. The
ap-
parent
K,
for the utilization process was
0.61
PM
with
a
V,,,
of
21.3 pmol/mg of proteinlmin. Oligomycin and
4,4’-diisothiocyanostilbene-2,2’disulfonic
acid were
inhibitory, whereas CMP-NeuAc, UDP-GlcNAc, aden-
osine 3‘-phosphate, 5’-phosphosulfate, atractylosides,
tunicamycin, 2’5’-ADP, and 3‘,5’-ADP were not,
showing that this transport process is distinct from
other nucleotide transporters previously described in
rat
liver Golgi. 75435% of the radioactivity incorpo-
rated
was
shown to be in 0-acetylated sialic acids, by
neuraminidase release, purification, and high pressure
liquid chromatography. The majority of the neuramin-
idase-resistant radioactivity was released by alkaline
hydroxylamine
as
[3H]acetylhydroxamate, but
a
sig-
nificant fraction was resistant to this treatment. The
nature of the non-sialic acid radioactivity remains un-
known. The existence of this transport mechanism pro-
vides yet another level
at
which the 0-acetylation of
sialic acids could be regulated.
The sialic acids are a family of
N-
and 0-substituted deriv-
*
This work was supported by United States Public Health Service
Grant
GM32373
from
the National Institutes
of
Health. The costs
of
publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked
“adver-
tisement”
in accordance with
18
U.S.C.
Section 1734 solely to indicate
this fact.
4
Faculty Fellow of the John
A.
and George L. Hartford Founda-
tion,
To
whom correspondence should be addressed.
atives of neuraminic acid
(1).
The elegant studies of Blix,
Klenk, Gottschalk, Schauer, and others have resulted in the
identification of more than
30
different free and glycosidically
bound sialic acids in nature
(1-3).
The most commonly found
sialic acid is N-acetylneuraminic acid (Neu5Ac).’ The other
sialic acids are all believed to
be
derived from this molecule
by different modifications and substitutions. The most com-
mon type of modification is the substitution of 0-acetyl esters
at
the hydroxyl groups of the 4-,
7-,
8-,
or
9-positions (2,
3).
These substitutions are known to have effects upon the action
of several enzymes
(4-lo),
upon alternate pathway comple-
ment activation
(ll),
and upon bacterial antigenicity and
pathogenicity
(12).
Earlier studies
of
the 0-acetylation reaction were carried
out in surviving slices of bovine submaxillary glands, using
[14C]acetate as a precursor. These studies, along with the
assay of an 0-acetyltransferase activity in crude homogenates
and fractions, were interpreted to indicate that the O-acety-
lation reaction could take place either upon bound or free
sialic acids
(13-15).
We have recently studied the O-acetyla-
tion reaction by pulse-chase experiments in tissue-culture cell
lines, and found that most, if not all, of the 0-acetylation
reaction takes place after the transfer of newly synthesized
sialic acids to glycoconjugates.’ In either model, it is necessary
to explain how the donor acetyl coenzyme
A
which
is
synthe-
sized in the
cytosol
gains access to the lumen of the Golgi
apparatus, where the sialylation reaction takes place. In the
case of the sugar nucleotides, Hirschberg, Sandhoff, and oth-
ers
(16-23)
have clearly shown evidence for the existence of
specific transporter proteins that mediate the entry of such
molecules into the Golgi apparatus. We postulated the exist-
ence of a similar carrier mechanism for acetyl-coA. In this
study, we demonstrate and characterize such an activity in
isolated rat liver Golgi vesicles, and show that 0-acetylated
sialic acids are the major (but not the sole) products. The
transport activity appears to be distinct from the others
previously described in the Golgi, and from that described by
Rome
et
al.
(24) in intact isolated lysosomes.
EXPERIMENTAL PROCEDURES
Materials-Most
of
the biological reagents used here were obtained
from Sigma. The following materials were obtained from the sources
The various sialic acids are designed by combinations of Neu
(neuraminic acid), Ac (acetyl), and Gc (glycolyl). The amino group at
the 5-position is always substituted with an acetyl (Ac) or a glycolyl
(Gc) group. Other substitution positions are indicated by numerals.
For example, N-glycolylneuraminic acid is written as Neu5Gc and
N-
acetyl-9-mono-O-acetylneuraminic acid as Neu5,9Acz (after Schauer
and others
(2)).
Other abbreviations used are: HPLC, high pressure
liquid chromatography; PAPS, adenosine
3’-phosphate,5’-phospho-
sulfate.
‘A.
Varki and
S.
Diaz, unpublished observations.
6600
This is an Open Access article under the CC BY license.
Acetyl-coA
Uptake
by
Golgi
Vesicles
6601
indicated Dowex 50 AG 1-X2 (H+ form) and Dowex 3-X4A (100-200
mesh, chloride form), Bio-Rad chemically synthesized Neu5Ac
(>99% purity), Kantoishi Pharmaceutical CO., Tokyo, Japan; Vibrio
cholerae and Arthrobacter ureafaciens neuraminidases, Calbiochem-
Behring;
[4-“C]N-acetylneuraminic
acid (56.8 mCi/mmol), [1-14C]
acetyl coenzyme A (53.5 Ci/mmol), [G-3H]coenzyme
A
(2.8 Ci/mmol),
[~ialic-9-~H]CMP-sialic acid (18.9 Ci/mmol), and [sialic-4,5,6,7,8,9-
“CICMP-sialic acid (247 mCi/mmol), New England Nuclear; [3H]
methoxyinulin (175 mCi/mg), [1,2-3H]deoxy-D-glucose
(28
Ci/mmol),
and [a~etyl-~HIacetyl coenzyme A (1.3 or 3.4 Ci/mmol), ICN Phar-
maceuticals Inc.; [l-“C]acetate (57 mCi/mmol) and UDP-[6-3H]
galactose (15.6 Ci/mmol), Amersham Corp.
Neuraminidase from Streptococcus sanguis was prepared as previ-
ously described (6). Authentic Neu5,9Ac2 was kindly provided by
Professor Roland Schauer, Christian Universitat, Kiel, Federal Re-
public of Germany. The Dowex 3-X4A resin was converted to the
formate form, as recommended by the manufacturer. All other chem-
icals were of reagent grade and were purchased from commercial
sources.
Isolation and Characterization of
Rat
Liver Golgi-Intact rat liver
Golgi vesicles were prepared by the method of Leelevathi (25). The
intactness and sidedness of Golgi vesicles were determined exactly as
described by Carey and Hirschberg (16). Galactosyltransferase (26-
28), glucose-6-phosphatase (29), and @-galactosidase (30) were mea-
sured as previously described. Protein was measured by the method
of Lowry
et
al.
(31).
The utilization of acetyl-coA by Golgi vesicles was assayed by
incubating 25-500 Wg of vesicle protein in 0.5-1 ml of TKM (10 mM
Tris-HC1, 150 mM KCl,
1
mM MgC12, pH 7.5) or PKM (10 mM
potassium phosphate, 150 mM KCI,
1
mM MgC12, pH 7.0) buffer at
22
“C in the presence of [“CI- or [3H]acetyl-CoA at various concen-
trations, as indicated. For some studies the reactions were quenched
with
4
ml of ice-cold buffer, the vesicles were re-isolated by centrif-
ugation, washed, and sonicated, and the perchloric acid-soluble and
-
insoluble radioactivity was determined exactly as described by Carey
and Hirschberg (16). Some of the studies were performed by directly
quenching the reactions with an equal volume of ice-cold
8%
perchlo-
ric acid and assaying only the acid-precipitable radioactivity. These
samples were incubated on ice for 15 min and spun at 10,000
X
g
for
10 min, the supernatant was removed, and the pellet was surface-
washed once with 4% perchloric acid. The pellet was then resuspended
in
1
ml
of
4% perchloric acid, re-isolated by centrifugation, and
dissolved in 700
p1
of
1
N
NaOH by heating at 55 “C for 15 min. The
dissolved material was then neutralized with 350
111
of
2
N
HCl, and
1
ml of the mixture was counted with 10 ml of Aquasol.
Chromatographic
Methods-Acylhydroxamates
were prepared by
alkaline hydroxylamine treatment, and separated by thin layer chro-
matography as described by Schauer (32). The chromatograms were
dried and cut into 0.5-cm strips, which were soaked in 0.5 ml of water
and 5 ml of Aquasol for determination of radioactivity. The radioac-
tive compounds were identified by comparison with an internal [“C]
acetylhydroxamate and other external nonradioactive hydroxamate
standards, which were prepared from the appropriate esters (32).
HPLC was used to separate acetyl coenzyme A, coenzyme A, and free
acetate by a modification of a previously described reversed-phase
ion-pair method (33). An Alltech RP-18 column (250
X
4.1 cm) was
eluted in the isocratic mode with 38% methanol, 0.5% tetrabutylam-
monium formate, pH 4.5, at
1
ml/min (System A). Under these
conditions, free acetate, CoA, and AcCoA elute at 2, 10, and 15 min,
respectively (not shown). The elution of unknown 3H-compounds was
compared with that of external standards (monitored at A254) and
internal [14C]acetate and [“Clacetyl-CoA standards. Various types of
sialic acids were separated from each other by HPLC on a Varian
Micropak AX-5 column eluted in the isocratic mode with acetoni-
trile:water:0.5
M
NaH2P0, (64:26:10) (System
B).
This method is
based on the ion suppression:amine absorption principle used for
separation of acidic oligosaccharides by Mellis and Baenziger (34).
As shown in Fig. 1, it is capable of separating a variety of mono- and
di-0-acetylated derivatives of N-acetylneuraminic and N-glycolyl-
neuraminic acid from each other. The elution of unknown radioactive
compounds was monitored in comparison to the nonradioactive
standards (detected by absorption at 200 nm) and to radioactive
standards obtained from metabolically labeled murine erythroleuke-
mia and myeloma cells.* In some cases, the sample was eluted at
1
ml/min with a linear gradient change to 54% acetonitrile, 36% water,
10% phosphate buffer over 20 min. The gradient gives somewhat
earlier elution times, but sharper resolution.
W
VJ
z
0
n-
W
VJ
a
5
0
I-
u
W
L
0
W
z
4
W
a
I I
5
10
TIME
(rnin)
FIG.
1.
HPLC
for the separation
of
various sialic acids.
Work was carried out with a Spectra-Physics 8700 Ternary Gradient
elution system on a Varian Micropak AX-5 column (30
X
0.4 cm).
The solvents used were acetonitrile (A), water (B), and 0.5
M
sodium
&hydrogen phosphate, pH 4.3 (C). The column was equilibrated in
64% A, 26%
B,
10% C and eluted in the isocratic mode; the sample
was loaded into a 50-111 loop. An example of such a separation is
shown here, using a mixture of sialic acids purified from bovine
submaxillary mucin; the elution was monitored at 200 nm, with the
chart speed at
1
cm/min. The position of known standards is as
indicated. INJ, injection peak.
Further
Fractionation
of
Labeled Golgi Vesicles-The vesicles were
washed in the same buffer used for the labeling and then sonicated
in
1
ml of 10 mM pyridinium formate buffer, pH 5.5, using 4
X
15-sec
pulses of a Heat Systems-Ultrasonics sonicator cell disruptor model
W-185-F with a probe setting of 30. The sonicate was centrifuged at
100,000
X
g for 30 min; the resulting pellet was called the “membrane”
fraction. It was washed once in
10
mM pyridinium formate, pH 5.5,
and resuspended in
1
ml of the same buffer. The 100,000
X
g
supernatant was adjusted to 90% absolute ice-cold ethanol and placed
at -20
”C
for
1
h. The flocculent precipitate was collected by centrif-
ugation at 1,500
x
g for 15 min and then resuspended in
1
ml of 5
mM pyridinium formate, pH 5.5. The ethanol supernatant was evap-
orated under reduced pressure and brought up in
1
ml of 10 mM
pyridinium formate, pH 5.5. All fractions were monitored for radio-
activity and stored at -20
“C
until future analysis.
Release, purification, and analytical de-0-acetylation of sialic acids
were carried out exactly as previously described (35) except for the
following changes: The addition of formic acid to the Dowex-50 eluate
was omitted; if the pH of the pooled Dowex-50 eluate was greater
than 3, it was applied directly to the Dowex 3-X4A column.
RESULTS
Characterization
of
Intactness and Purity
of
Golgi Vesicles-
Rat liver Golgi vesicles were isolated and characterized as
described under “Experimental Procedures.”
A
typical prep-
aration (used for many of the studies reported here) was
enriched 103-fold for galactosyltransferase (61% yield), 2.3-
fold for glucose-6-phosphatase (1.4% yield), and 2.2-fold for
@-galactosidase (1.3% yield). The intactness of the vesicles
was judged by their latency toward neuraminidase-catalyzed
removal of labeled sialic acid exactly
as
described by Carey
and Hirschberg (16). By this criterion, greater than
90%
of
6602
Acetyl-coA
Uptake
by
Golgi Vesicles
the vesicles were sealed and of the correct topographic orien-
tation, in all Golgi preparations used in this study.
The
Incorporation
of
Radioactivity from PHJAcetyl-CoA
into Rat Liver Golgi Vesicles-The intact, right-sided liver
Golgi vesicles were incubated with [3H]acetyl-CoA in TKM
buffer as described under “Experimental Procedures.”
As
shown in Fig. 2, these vesicles incorporated acid-soluble and
acid-insoluble radioactivity from [3H]acetyl-CoA in a manner
dependent upon time, temperature, protein concentration,
and acetyl-coA concentration. The reaction was not linear
with time beyond
5
min (detailed data not shown). The
incorporation at
0
“C (which could include any occurring
during the subsequent
4
“C centrifugation step) ranged from
20
to
40%
of that at room temperature in various experiments.
Under all conditions studied, the acid-soluble radioactivity
closely paralleled the acid insoluble component and repre-
sented from 25 to
40%
of the pellet-associated radioactivity.
The incorporation of radioactivity seemed to approach satu-
ration with the addition of increasing amounts of radioactive
acetyl-coA in the 3-5
p~
range. Addition of an excess of
nonradioactive acetyl-coA (at
40
p~)
markedly reduced the
incorporation of radioactivity (see Fig.
2).
These findings
suggested the existence of a specific mechanism for the utili-
zation of acetyl-coA by the Golgi vesicles.
Evidence
for
Accumulation and Concentration
of
Radioactive
Compounds within the Vesicles-If an active transport process
were involved in these phenomena, the radioactivity should
show
a
relative concentration in the vesicles over that in the
medium. Hirschberg and others (17, 20) have previously
shown that impermeant
(
[3H]methoxyinulin) and permeant
([1,2-3H]deoxyglucose) markers can be used to accurately
estimate the mean total pellet volume,
[
V,], of Golgi vesicles,
the volume outside the pellet,
[
V,], and thus the internal
volume of the pellet, [Vi]
(=
[V,]
-
[V,]).
This information
was
compared with that of the incorporation of various radio-
actively labeled nucleotide sugars under identical conditions
to
obtain a “penetration index” in each case. This then
allowed a determination of the fold concentration of the label
in the vesicles achieved by the transport system in question.
We used the identical approach to ask if the Golgi vesicles
were indeed concentrating the label from [3H]acetyl-CoA. The
details of the experiment are described in Table
I.
Briefly,
when Golgi vesicles were incubated in TKM buffer for 20 min
at 22 “C with [3H]acetyl-CoA at
1.54
p~,
the total of radio-
active solutes in the pellet was 86.9 pmol/mg of the radioac-
tivity. Based upon the [Vi] value (0.26 pl/pellet volume/mg of
Golgi protein) obtained under identical conditions, the con-
centration of radioactivity inside the pellet was calculated to
be 187.1
pM.
This represents a 121-fold concentration of the
label within the vesicles over that in the medium. This is very
similar to the findings of Hirschberg and others (17, 20) for
other nucleotides such as CMP-sialic acid, GDP-fucose, and
PAPS and suggests that a similar active transport mechanism
is present for acetyl-coA in Golgi vesicles.
Characterization
of
Intravesicular
Law
Molecular Weight
Contents-As described above, the intravesicular low molec-
ular weight (acid-soluble) radioactivity represented from 25
to
40%
of the total radioactivity incorporated and closely
correlated with the acid-insoluble fraction under various con-
ditions. We analyzed this fraction further by HPLC in System
A
as described under “Experimental Procedures.” We found
that greater than 99% of the label was in the form of free
acetate. We next studied the radioactivity remaining in the
medium after a 20-min incubation with
1
mg
of
Golgi vesicles
in TKM buffer. We found that
60%
of this label was now also
in the form of free acetate. An identical incubation in the
absence of Golgi vesicles resulted in only
4%
breakdown of
the acetyl-coA. This rapid breakdown of the added acetyl-
CoA was thus being catalyzed by the presence of the Golgi
vesicles. This also raised the possibility that the incorporation
of label observed was actually that of free acetate rather than
the acetyl-coA molecule.
Free Acetate
Is
Not Taken up
by
Golgi Vesicles-To inves-
tigate this possibility we incubated Golgi vesicles with a
I*
PROTEIN
(mg)
C
1
2
3
4
ACETYL.CoA
(PM)
FIG.
2.
Incorporation of radioactivity into isolated rat liver Golgi vesicles from [~cetyl-~H]acetyl-
CoA.
Golgi vesicles were preincubated in TKM for
30
s,
in the presence
or
absence of ATP
(0.5
mM final
concentration). The reaction was started by the addition of varying amounts
of
[3H]acetyl-CoA, and the incubation
was conducted under varying conditions of temperature, time, and protein concentration. The reaction mixtures
were then centrifuged at
100,000
X
g
for
30
min at
4
“C, and the pellet-associated radioactivity (acid-soluble and
under the following conditions: 22 “C, +ATP
(0,O);
22 “C, -ATP
(A,
A);
0
“C, +ATP
(0,
W);
and 22 “C, +ATP,
acid-insoluble) was determined as described under “Experimental Procedures.” The various points show incubations
+40
PM
unlabeled acetyl-coA
(*).
The open symbols represent acid-insoluble radioactivity, and the closed symbols
represent acid-soluble radioactivity in each case. The
asterisk
represents total pellet-associated radioactivity. The
studies in
panels
A
and
C
were carried out with
1
mg of Golgi protein; those in
panels
A
and
B
were carried out
with [3H]acetyl-CoA at
0.9
phf;
those in
panels
B
and
C
were carried out for
10
min at the temperatures indicated.
Acetyl-coA Uptake
by
Golgi
Vesicles
6603
TABLE
I
Evidence for translocation
of
['Hlacetyl-CoA
into
Golgi
vesicles:
concentration of radioactive solutes within the vesicles
Golgi vesicles
(0.25
mg of protein) were incubated with [acetyL3H]
acetyl-coA
(1.54
nmol,
4.4
X
lo6
dpm) at
22
'C for
10
min in a final
volume of
1
ml of TKM buffer, containing
0.5
mM ATP. Parallel
identical incubations were made with [SH]inulin and [3H]deoxyglu-
cose. The vesicles were then chilled and re-isolated, and the radioac-
tive solutes were determined as described under "Experimental
Pro-
cedures." Mean values of duplicate determinations were used to
calculate the following parameters exactly as described elsewhere by
Hirschberg and others
(17,20).
[S,]
is the concentraton of the acetyl-
CoA in the media;
[
V,] is the total volume
of
the pellet determined
from the deoxyglucose control;
[
V.]
is the volume trapped outside the
pellet, and is determined from the inulin control;
[Vi]
is the volume
inside the pellet determined by subtracting the two former values;
[S,]
is
the total radioactive solute in the entire pellet;
[So]
is the
solute outside the vesicles; and [Si] is the solute present inside the
vesicles.
Radioactive solutes in Volume of
Golgi
[
S"1
pellet pellet
[si1
[Si1
-
[
Sml
st
so
si
v, v.
vi
p~
pmollmg
protein
pl/mg
protein
1.54
86.8
38.2 48.6 0.70 0.44
0.26
186.9 121.4
TABLE
I1
Uptake and incorporation
of
&led
molecules
into
Golgi
vesicles
Rat liver Golgi vesicles
(1
mg of protein) were incubated at
22
"C
in TKM buffer with
0.5
mM ATP and the radiolabeled compounds at
the concentrations indicated. After
20
min,
3
ml of ice-cold TKM
buffer was added, the vesicles were reisolated, and radioactivity was
determined.
Radioactivity found in:
Radioactive Final
compounds concen-
~d~
Pellet-associated
added tration (starting) Acid- Acid-
soluble insoluble
*M
4m
[3H]Acetyl-CoA
1.0 3,052,398 124,111 196,628
[1-"CIAcetate
5.0 696,302 561 89
(3H/14C Ratio)
(4.4) (221) (2209)
["CIAcetyl-CoA
3.0 1,061,040 46,985 73,950
A~etyl-[~H]CoA
0.2 414,960 11,387 8,584
(3H/14C Ratio)
(0.391) (0.242)
(0.116)
[3H]Coenzyme ASH
0.2 1,283,632 106,638 145,458
(W
media dpm)
(100) (8.3) (11.3)
[3H]Deoxyglucose
0.15 11,676,578 8,260 766
(%
media dpm)
(100) (0.07)
(0.006)
mixture of [14C]acetate and [3H]acetyl-CoA.
As
shown in
Table
11,
in spite of
a
5-fold molar excess of the free acetate,
the I4C label was taken up at less than
1%
of the rate seen for
the 3H label. This shows that although free acetate was found
both outside and inside the vesicles at the end of the incuba-
tion, the intact acetyl-coA molecule was involved in the
incorporation seen. This also shows that significant conver-
sion of free acetate
to
acetyl-coA is not taking place in the
presence of the added
ATP.
Evidence
for
Uptake
of
the Entire Acetyl Coenzyme A
Mol-
ecule-To confirm that the entire acetyl-coA molecule was
being taken up, we compared the uptake of simultaneously
added [14C]acetyl-CoA with a~etyl-[G-~H]CoA. The acetyl-
[G-3H]CoA was prepared by acetylation of [G-'H]CoASH by
treatment with acetic anhydride, as described by Stadtman
(36).
As
shown in Table
11,
we found that the 'H/14C ratio of
the intravesicular acid-soluble fraction was very close
to
that
of the starting media, strongly suggesting that both the acetyl
and the
CoA
portions of the molecule were being transported.
Surprisingly, we found that 3H radioactivity from the [G-3H]
coenzyme A portion of the molecule was also being incorpo-
rated into acid-insoluble materials
at
a significant rate.
As
a
"control" we had also incubated the Golgi vesicles with [3H]
CoA.
In
this case, we found that there was again
a
considerable
concentration of the label in the vesicles relative to the t3H]
deoxyglucose control marker (see Table
II),
suggesting
vec-
torial transport of coenzyme ASH. We are uncertain of the
nature of the radioactivity incorporated from the t3H]CoA
into acid-insoluble materials. Since this labeled compound
was originally prepared by 3H gas exchange into all available
sites, much further work must be done to confirm the signif-
icance of this finding.
Nonionic Detergents Abolish Uptake
and
Utilization-As
shown in detail below, [3H]O-acetylsialic acids were found to
be
a
major component of the acid-insoluble fraction. These
products are presumably formed by the action of O-acetyl-
transferases on endogenous acceptors, utilizing the [3H]ace-
tyl-CoA as
a
donor. Thus, the incorporation of radioactivity
into the acid-insoluble materials could be limited by at least
two factors: the rate of transport of the donor molecule into
the vesicles or the activity of the 0-acetyltransferase(s). To
differentiate between these two possibilities, we investigated
the effects of the nonionic detergent Triton X-100 on the
incorporation of radioactivity into acid-insoluble materials.
As
demonstrated in Fig.
3,
we found that the detergent was
without effect until the concentration approached that of its
critical micellar concentration (0.015%, see Ref.
37).
Above
this level, where the acetyl-coA should be freely accessible to
the 0-acetyltransferases inside the lumen, the incorporation
was markedly diminished.
As
a positive control, we showed
(see Fig. 3) that the activity of the Golgi enzyme galactosyl-
transferase toward an
emgeenous
acceptor (free GlcNAc at 100
m~)
was greatly enhanced above the critical micellar concen-
tration of the detergent. The most likely explanation for these
findings is that at the concentration
of
acetyl-coA used (1.3
IM)
the rate-limiting step was the transport process rather
0
001
0
01
01
TRITON
X-100
CONCENTRATION
1%)
I
FIG.
3.
Effects
of
nonionic detergent
on
Golgi
utilization
of
[SH]acetyl-CoA
and galactosyltransferase activity.
For ['HI
acetyl-coA utilization, Golgi vesicles were preincubated in TKM
buffer with Triton
X-100
at the concentrations indicated for
30
s.
The label
(0.9
PM
find concentration) was added, the reaction was
quenched after 6 min with perchloric acid, and the acid-insoluble
radioactivity was determined as described under "Experimental Pro-
cedures."
For
determination of the activity of galactosyltransferase
toward the exogenous acceptor GlcNAc, the reaction waa started by
addition of the enzyme
to
a tube containing all other elements of the
assay. The transfer of label from UDP-[6-3H]galactose to GlcNAc
was determined exactly as described elsewhere
(27).
under conditions
that were linear with time and protein concentration.
6604
Acetyl-coA Uptake by Golgi Vesicles
than the 0-acetyltransferase activities. To rule out the pos-
sibility that the detergent was inactivating the enzyme(s)
above its critical micellar concentration, we also studied the
effects of increasing the acetyl-coA concentration. As shown
in Fig. 4, the incorporation of acetate into acid-insoluble
material was saturable with acetyl-coA in the 5-10
p~
range
with intact Golgi vesicles. In the presence of
0.2%
Triton
X-
100, significant activity did eventually appear in the 10-100
p~
range. This suggests that the 0-acetyltransferases were
still intact, but could show significant activity only at much
higher concentrations of the donor molecule. This is in keep-
ing with previous studies of an 0-acetyltransferase activity in
submaxillary glands, which had a
K,,,
for acetyl-coA of 100
p~
(15). The only other relatively unlikely explanation of
these findings is that the detergent could be modifying the
K,
of the enzyme itself.
Optimization of Conditions for Further Studies-Further
studies of the kinetics and requirements of the reaction were
greatly hampered by the very significant activity seen
at
0
“C
(see above), which presumably also represented uptake occur-
ring during the subsequent 4 “C centrifugation step. Not
surprisingly, this “background activity was highly variable,
depending upon the exact handling conditions of each sample.
This problem was further compounded by our finding (see
below) that the Golgi vesicles contain an endogenous esterase
activity. These facts, coupled with the lack of linearity of the
assay beyond very short time points, made it very difficult to
obtain valid blanks and accurate time points using the cen-
trifugation method. We therefore carried out all further stud-
ies of the reaction by directly quenching the reactions with
perchloric acid and directly determining the acid-precipitable
radioactivity. This eliminated analysis of the acid-soluble
radioactivity within the vesicles. However,
as
indicated above,
in all cases studied by centrifugation, the radioactivity in the
acid-soluble fraction closely paralleled that in the insoluble
fraction. Furthermore, the detergent experiments described
above strongly suggest that the intactness of the vesicles (and
therefore the transport process) is the rate-limiting factor in
the incorporation of radioactivity into acid-insoluble mate-
rials. Thus, the utilization of radioactivity by the vesicles
under various conditions is probably an indirect measure of
the transport process itself.
ACETYL CoA
(pM)
FIG.
4.
Effects of increasing acetyl-coA concentration in
the presence and absence
of
detergent.
Golgi vesicles were prein-
cubated for 30
s
in TKM buffer in the presence
or
absence of
0.2%
Triton
X-100
(final concentration). The reaction was started by
addition of [3H]acetyl-CoA at the various concentrations indicated.
The reactions were quenched after 6 min with perchloric acid, and
the acid-insoluble radioactivity was determined as described under
“Experimental Procedures.” The incorporation of acetate was calcu-
lated based upon the specific activity of the added label in each case.
Using this approach, we studied the effects of various
buffers, salts, divalent cations, pH values, and other agents
on the process during 3-5-min incubations. Maximal activity
was seen
at
pH
7.0
in the presence of potassium phosphate
buffer (20 mM) and potassium chloride
(150
mM). Other
attempts to manipulate the buffer composition to more closely
mimic the normal composition of natural cytosolic salts (38)
(uiz.
addition of sulfate, increase
of
phosphate with lowering
of chloride) did not improve the activity. Under the optimal
buffer conditions and in the presence of ATP, the linearity of
the assay was greatly improved (completely linear up to
8
min, data not shown). Using these optimal salt and buffer
concentrations, the effects of other agents were studied. The
addition of 2-mercaptoethanol
(a
reducing agent), 2,3-dimer-
captopropanol (a reducing agent and zinc chelator which
inhibits pyrophosphatases) (39), sodium fluoride (an inhibitor
of pyrophosphatases) (40, 41), or bovine serum albumin
(1
mg/ml) removal of divalent cations from the buffer, substi-
tution of Ca2+ for
M$+,
or addition of 2 mM EDTA were
without remarkable effect on the process.
Effects of Substrate Analogues
and
Other Compounds-The
studies
so
far suggested that the intact acetyl-coA molecule
was involved in the utilization process. We further explored
the specificity of this reaction by studying the effects of
various substrate analogues and other compounds known to
be taken up by rat liver Golgi vesicles. As shown in Table 111,
the utilization process was markedly inhibited by coenzyme
ASH, butyryl-CoA, and palmitoyl-CoA in concentrations in
the
1-5
PM
range. These concentrations are well below the
critical micellar concentration of these molecules (42, 43),
TABLE
I11
Effects of substrate analogues and other compounds on
the
incorporation of [3Hlacetyl-CoA into rat liver
Golgi
vesicles
Rat liver Golgi vesicles
(215
pg
of protein) were preincubated in
500
p1
of PKM buffer with
1
mM
ATP
and the various compounds at
the concentrations indicated for
3
min at
22
“C. [3H]Acetyl-CoA
(1
pCi) was added, and the reactions were quenched after
6
min with
500
p1
of
8%
perchloric acid. The acid-precipitable radioactivity was
then determined exactly as described in the legend to Table
I1
and
expressed as a percentage of that obtained in a control incubation
with no additions. Some of the compounds were added as solutions
in absolute ethanol. The final concentration of ethanol never ex-
ceeded
0.5%
in any incubation. This concentration
of
ethanol had no
significant effects on the reactions (not shown). DIDS, 4,4’-diisothio-
cyanostilbene-2,2’-disulfonic
acid.
%
of
control activity at concentration
Compound
0.1pM
1
pM
5pM
25”
100pM
Coenzyme ASH 87 55 31
8
3
Butyryl-CoA 89 73 58 14
Palmitoyl-CoA 90 59 35 3
CMP-Neu5Ac 85 80 90 91
Atractyloside 74 85 89
DIDS
Carboxylatractyloside 87 78 91
PAPS
75 54 13
2’,5’-ADP 94 94
81
UDP-GlcNAc 85 83 90 77
87
105
78
3’.5”ADP 95 90 96
%
of
control activitv
Tunicamycin
(0.1 pg/ml)
(1.0 dml)
(5.0 pg/ml)
Oligomycin (50 gg/ml)
Ammonium sulfate (10 mM)
Monensin
(2
pM)
Sodium vanadate (0.1 mM)
N-Ethylmaleimide
(0.2
mM)
91
88
88
56
84
96
89
95
Acetyl-CoA Uptake by Golgi Vesicles
6605
implying that the inhibition is not related to
a
detergent
effect. This suggests that the coenzyme A moiety is recognized
during the process and supports the findings with the free
acetate and double-labeled compounds, described above. Pre-
vious studies have indicated the existence of independent
transport mechanisms for several other nucleotides in rat
liver Golgi vesicles. As shown in Table
111,
we found that
CMP-sialic acid (16, 17) and UDP-GlcNAc (22) were without
significant effect upon the acetyl-coA utilization. Tunica-
mycin, which inhibits UDP-galactose transport by Golgi ves-
icles (18,19), was also without effect. The sulfate donor PAPS
is similar to acetyl-coA in two ways. First, both molecules
contain a 3’,5‘-ADP group, and second, palmitoyl-CoA has
been previously shown to be
a
good inhibitor of PAPS uptake
by rat liver Golgi (21). However,
as
shown in Table
111,
the
acetyl-coA utilization process under study here is quite dif-
ferent in other respects from the PAPS system. First, coen-
zyme A was not found to affect PAPS uptake (X), while
3’,5’-ADP, atractyloside, and carboxyatractyloside, which in-
hibit PAPS uptake (21), were without effect on the acetyl-
CoA uptake. Second, PAPS itself had little effect in concen-
trations well above its previously reported
K,,,
of
0.7
pM
(20).
Besides the inhibition by palmitoyl-CoA, the only other sim-
ilarity between the two processes was the inhibition by the
nonspecific anion transport inhibitor 4,4-diisothiocyanostil-
bene-2,2’ disulfonic acid (21). These findings show that the
acetyl-coA uptake process described here is different from all
previously described Golgi uptake mechanisms.
Further Investigation of the ATP Effect-With the use of
the improved buffer and pH conditions, the ATP effect be-
came less pronounced. A maximum of 28% enhancement was
seen
at
0.5 mM ATP in
PKM
buffer
at
22 “C. Higher concen-
trations of ATP had no further effect; in fact, concentrations
above 3 mM caused inhibition. The addition of an ATP-
regenerating system (creatine phosphate at 1.2 mM and crea-
tine phosphokinase
at
0.125 mg (150 units/ml)) also did not
improve the ATP effect, further suggesting that ATP break-
down was not the limiting factor in the ATP effect. The
uptake
at
0
“C, which was 37% of that
at
22 “C in this
experiment, was unaffected by ATP. Preincubation of the
Golgi vesicles in 0.5 mM ATP for 10 min prior to the addition
of the acetyl-coA did not provide any improvement over the
effect of simultaneous addition.
The
Effects of ATP Are Not Related to the Golgi “Proton
Pump”-Recent studies have demonstrated the existence of
a Golgi proton pump that utilizes ATP or GTP in the presence
of Mg+, to maintain an acidic pH in the interior of the Golgi
vesicles (44, 45). The lack of dependence of the ATP effect
on M$+ already suggested that it was not due to enhancement
of the ATPase function. To confirm this, we demonstrated
(see Table
111)
that N-ethylmaleimide, which inhibits the
Golgi proton pump (45), and agents such as ammonium sulfate
and monensin, which would collapse
a
pH gradient, were
without significant effect on the process. Unexpectedly, oli-
gomycin, which affects the mitochondrial Mg2”ATPase and
should not affect the Golgi proton pump (45), was capable of
inhibiting the acetyl-coA utilization process. Sodium vana-
date, which affects certain other ATPases, was without effect.
Kinetics of the Uptake Process-Because of the difficulties
with obtaining true blanks under various conditions (see
above), an accurate study of the kinetics of the uptake process
was not possible. Therefore, we studied the kinetics of incor-
poration of the radioactivity from [3H]acetyl-CoA into acid-
insoluble materials alone. As shown in Fig. 5, this gave an
apparent K,,, of 0.61
p~,
with
a
V,,, of 21.73 pmol/mg/min.
Since our earlier studies had suggested that the rate-limiting
02468
I1
/SI
FIG.
5.
Kinetics
of
incorporation
of
label
from
[~cetyZ-~H]
acetyl-coA into rat liver Golgi vesicles.
Golgi
vesicles
were
preincubated
for
30
s
at
22
“C
in
PKM
buffer.
The reactions
were
started
by addition
of
[3H]acetyl-CoA
in
various amounts
for
the
final concentrations indicated. The
reactions
were quenched
after
5
min
by
addition
of
perchloric
acid,
and
the
acid-insoluble radioactivity
was
determined
as
described
under
“Experimental Procedures.” All
points shown
are
the
mean
of
duplicate determinations. The
data
are
presented
as
a
function
of
[
v]
uersw
[SI
and
as
a double-reciprocal
plot. The latter
was
used
to
calculate
an
apparent
K,,,
of
0.6
PM
and
a
V,.
of
21.73 pmol/mg/min.
factor in the accumulation of the acid-insoluble radioactivity
was probably the transport process itself, the
K,
value ob-
tained here is probably
a
reasonable approximation of the
kinetics of the transport process. More accurate studies must
await the purification of the transport protein itself.
Stability-The activity was markedly unstable at 37 “C.
About 40% of the activity was lost upon preincubation at
room temperature for 10 min, regardless of whether ATP was
present. A slower but significant loss of activity occurred even
at
0
“C. The activity appeared stable when the vesicles were
gently suspended into
10
mg/ml bovine serum albumin (with
a
Dounce homogenizer) and stored in liquid nitrogen for up
to
2
weeks. However, prolonged storage under these conditions
also resulted in gradual loss of activity. A single cycle of
repelleting and resuspension caused complete loss of activity.
Thus, it was not possible to carry out some types of useful
experiments (e.g. pretreatment with Pronase) (17).
Characterization
of
Macromolecular Products-We next
characterized the nature of the products formed within the
Golgi vesicles during the incubation with [3H]acetyl-CoA. A
single large preparation of Golgi vesicles was made in PKM
buffer for 30 min. The labeled vesicles were pelleted, washed,
and disrupted by sonication in hypotonic buffer, and the
membranes were reisolated by centrifugation, as described
under “Experimental Procedures.” The labeled membranes
thus obtained contained
60%
of the total radioactivity origi-
nally found in the vesicles. The soluble macromolecular prod-
ucts from the supernatant were precipitated with
90%
cold
ethanol and contained 10% of the total label, leaving
30%
of
the total label in the ethanol-soluble fraction. For the follow-
ing characterization
of
the “macromolecular” fraction, the
membranes and the resolubilized ethanol precipitate were
then pooled together.
Our original prediction of the existence of this transport
mechanism was based upon the expectation that it would
make the donor available for a Golgi-localized sialic acid-
specific 0-acetyltransferase. We therefore first looked for the
presence of [3H]-O-acetylsialic acids in the membrane-bound
fraction. An aliquot of the labeled macromolecular fraction
6606
Acetyl-coA
Uptake
by
Gob
Vesicles
was treated with a mixture of neuraminidases from
S.
sanguis,
V.
cholerae,
and
A.
ureafaciens (35), which would release most
known sialic acids. The incubation conditions were as previ-
ously described (35), except for the addition of
0.2%
Triton
X-100,
to ensure maximal exposure of all macromolecules to
the enzymes. In a parallel control experiment, these condi-
tions were shown to release greater than 90% of the label
incorporated into Golgi vesicles from CMP-["CINeuNAc.
This treatment resulted
in
release of 75-85% of 3H label from
[3H]acetyl-CoA-labeled membranes in different experiments.
The released label was further purified using a previously
described method (35) which gives an 80-90% yield of sialic
acids with less than
5%
loss of 0-acetylation. When an aliquot
of the purified label was treated with alkaline hydroxylamine
and studied by TLC, almost all of the radioactivity co-mi-
grated with ['4C]acetylhydroxamate, and away from lactyl-,
glycolyl-, butyryl-, and palmitoylhydroxamates (not shown).
This suggests that the incorporated label was primarily in
0-
acetyl esters, and that other acyl groups were not being
formed. Another aliquot of the purified label was studied by
a HPLC method which separates the different types
of
sialic
acids.
As
shown in Fig. 6, all of the label was retained by the
HPLC column and the major peaks eluted in positions char-
acteristic for mono- and di-0-acetylated sialic acids (compare
with Fig.
1).
As expected, none of the peaks eluted in the
position of non-0-acetylated Neu5Ac or Neu5Gc. All of the
label could be converted into free acetate (see lower panel
of
Fig. 6) by treatment with base under conditions known
to
de-
0-acetylate sialic acids. In all, there were at least eight such
distinct base-labile peaks. Since all of these compounds were
released by neuraminidase and copurified through several
1
n
e
E
x
b
V
-
I
FRACTION
(ml)
FIG.
6.
HPLC
for the separation of various sialic acids.
Work
was carried out exactly as described in the legend
to
Fig.
1.
The
upper
panel
shows the separation
of
sialic acids purified from
the macromolecular fraction
of
Golgi
vesicles labeled with
[acetyL3H]
acetyl-coA, as described in the text. The
lower
panel
shows an
identical aliquot subjected to de-0-acetylation with
0.1
N
NaOH on
ice
for
45
min. In each case,
an
internal standard of ["CC]NeuSAc was
added. The position
of
elution
of
free acetate and
of
other standard
sialic acids is
as
indicated (compare with Fig.
1).
steps with sialic acids, it is reasonable
to
assume that they
are all 0-acetylated sialic acids. They could represent various
0-acetylated derivatives of endogenous Neu5Ac and Neu5Gc
and also of other endogenous sialic acids that were already
substituted with other groups (e.g. methyl groups)
(2).
Since
the label is exclusively in the 0-acetyl groups, it is very
difficult
to
characterize these compounds further.
We therefore tried alternative approaches to their further
identification. First, an aliquot of the purified labeled material
was incubated for 14 h at pH 7.5 at 37 "C. Under these
conditions, migration of 0-acetyl esters along the exocyclic
side chain of sialic acids, from the 7- and 8-positions to the
9-position, is known
to
occur
(2,
35), while no significant de-
0-acetylation occurs? When the incubated label was then
reapplied to the HPLC column, there was indeed very little
evidence of de-0-acetylation (formation of free acetate). How-
ever, the profile and relative heights
of
the various peaks had
changed significantly, strongly suggesting that migration of
0-acetyl esters had occurred (not shown). This suggests that
some of the peaks are isomers of each other with 0-acetyl
groups at different locations on the 7/8/9-side chain.
We also carried out an identical purification from Golgi
vesicles labeled with CMP-[14C]Neu5Ac in the presence
of
[3H]acetyl-CoA, or CMP-[9-3H]sialic acid in the presence
of
[14C]acetyl-CoA. In such preparations, a portion of the sialic
acid label (34%) did migrate in the positions of several of
the peaks seen with the [3H]acetyl-CoA preparation (data not
shown). In the case of the sialic acids labeled with the CMP-
[9-3H]sialic acid, all of the label should
be
in the 9-position
of the molecules. Treatment of [9-3H]sialic acid with mild
periodate at neutral pH (46-48) resulted in complete conver-
sion of the
3H
label into [3H]formaldehyde, which can be
removed by evaporation? However, if the sialic acids are
substituted at the
9-
or 8-positions with 0-acetyl esters, the
periodate reaction would be blocked
(11,
49). When such
periodate treatment was carried out on the purified sialic
acids from the CMP-[9-3H]sialic acid labeling, most of the
label indeed became volatile. When the remaining nonvolatile
radioactivity was reapplied
to
the HPLC column, it
was
found
that the major non-0-acetylated [9-3H]Neu5Ac peak
was
com-
pletely destroyed, while many of the peaks corresponding
to
the 0-acetylated derivatives had survived the treatment (not
shown). This further suggests that the labeled compounds are
indeed 0-acetylated sialic acids. However, these double-la-
beled compounds represented only 34% of the total labeled
sialic acids, making it difficult
to
use them for further defin-
itive identification. Another approach we tried was to release
and purify the endogenous sialic acids of unlabeled Golgi
vesicles. However, the amount of these endogenous sialic acids
was very small (4.6 nmol/mg of protein). Thus, the amount
of unlabeled sialic acids we obtained
was
also inadequate
to
characterize them completely by conventional methods.
Thus, although definitive identification
was
not possible,
the various radioactive peaks seen on the HPLC analysis are
very likely
to
be 0-acetylated sialic acids. First, they were
released by neuraminidase from the macromolecular fraction
of the labeled Golgi vesicles. Second, they copurified with
sialic acids through several different steps. Third, they eluted
from the AX-5 HPLC column under conditions that are very
characteristic for sialic acids. Last, all of the label could
be
converted to free acetate by base treatment, and
to
acetylhy-
droxamate by alkaline hydroxylamine treatment.
Evidence
for
a
Golgi
0-Acetyl-esterase
and
an Acetyl-CoA
Hydrolase-During the release and purification
of
the
13H]O-
acetylated sialic acids, we noted that the final recovery was
only 40-50% of the label that was apparently released by
Acetyl-coA
Uptake
by
Golgi
Vesicles
6607
neuraminidase. Under identical conditions, the recovery of
14C-labeled sialic acids
was
80-90%.
This discrepancy was
found to be due
to
the presence of free acetate after the
neuraminidase incubation. The [3H]acetate was then being
lost as [3H]acetic acid during the drying-down step after
Dowex-50 chromatography. By incubating vesicles prelabeled
with [3H]acetyl-CoA or unlabeled vesicles with the [3H]O-
acetylsialic acids, we have found evidence for an esterase
activity intrinsic to the Golgi membranes that is capable of
cleaving 0-acetyl groups from sialic acids? The activity of
this enzyme during the neuraminidase incubation probably
explains the losses of label seen.
As
predicted by the studies
described above, we have also found evidence for an acetyl-
CoA hydrolase in the Golgi vesicles that can explain the
extensive breakdown of the labeled acetyl-coA during the
incubations. Further studies of both activities are under way.
Characterization
of
Neuraminidase-resistant Radioactiu-
ity-The 15-25% of radioactivity in membranes that re-
mained resistant to neuraminidase release was characterized
further as follows. Only
10%
of this label was released by
neutral hydroxylamine treatment. With alkaline hydroxyl-
amine treatment (which should release all 0-acyl esters), only
72% of this label was released, even with repeated treatments.
The product of these treatments co-migrated with acetylhy-
droxamate on TLC. This leaves 28% of this neuraminidase-
resistant fraction resistant to alkaline hydroxylamine (or 4-
7% of the original total radioactivity). We are uncertain of
the nature
of
these various components of neuraminidase-
resistant radioactivity. The neutral hydroxylamine-sensitive
component could represent thiol esters. The akaline-hydrox-
ylamine sensitive component could represent 0-acetyl esters
on neuraminidase-resistant sialic acids or on other molecules.
The hydroxylamine-resistant fraction could be in other link-
ages, such as N-acetyl groups of proteins or sugars. These
findings raise the possibility that the acetyl-coA transport
into the Golgi apparatus serves functions other than the
0-
acetylation of sialic acids.
DISCUSSION
In this study we have demonstrated evidence for a rat liver
Golgi acetyl-coA translocator that provides the donor for
acetylation reactions within the Golgi apparatus. Surpris-
ingly, the transporter also concentrates coenzyme ASH in the
vesicles for some other unidentified donation reaction. Since
CoA itself can be transported into the Golgi vesicles, it is
impossible to conclusively demonstrate that the entire acetyl-
CoA molecule is indeed being transported. However, it ap-
pears most likely that this is the case. The results suggest
that upon entry into the Golgi lumen, the acetyl-coA molecule
is either utilized for transfer of acetyl groups, or is immedi-
ately degraded to free acetate.
The characteristics of the activity described here make it
quite distinct from the acetyl-coA utilization by rat liver
lysosomes described by Rome et al. (24). In that case, there is
no evidence for translocation of the entire molecule. The
acetyl group is donated primarily to the free amino group of
terminal a-linked glucosamine residues that appear during
the degradation of heparan sulfate proteoglycan. Further-
more, the lysosomal process is very poorly inhibited by coen-
zyme ASH and is much more stimulated by
ATP
(24).
By several criteria this transporter also appears to be dis-
tinct from all others described to date in the Golgi apparatus
(16-23, 50), including that responsible for the translocation
of the structurally related molecule
PAPS
(20, 21, 50). In
these previous studies, Pronase digestion was used to show
that the transport processes in question were mediated by
proteins which, at least in part, faced the exterior of the Golgi
vesicles (17, 20). We were unable to carry out similar experi-
ments because of the extreme lability of the acetyl-coA uti-
lization process (see “Results”). These previous studies also
looked at the subcellular distribution of the sugar nucleotide
transport systems and demonstrated a Golgi localization for
the activities. However, unlike the case with sugar nucleotides,
acetyl-coA is known to be utilized by other organelles such
as lysosomes (24) for purposes other than the 0-acetylation
of sialic acids. We therefore did not study the relative enrich-
ment of this activity in the Golgi vesicles.
The major products found in the macromolecular fraction
were 0-acetylated sialic acids. Our data do not allow us to
clearly distinguish whether the translocator function is due
to a completely distinct protein from the sialic acid O-acetyl-
transferase(s). However, the fact that other (unidentified)
acetylated products could be demonstrated makes this likely
to be the case. It should be noted that this transport process
could have a very important role in concentrating the acetyl-
CoA donor to a level sufficient to drive the 0-acetylation
reaction.
A
previous study of a sialic acid-specific O-acetyl-
transferase in bovine submaxillary glands (15) found an ap-
parent
K,,,
of 100
PM.
For example, cytosolic levels
of
acetyl-
CoA that have been measured in brain tissue range from
2
to
6
PM
(51). Since 0-acetylation of sialic acids clearly occurs in
brain tissue (2), the concentrating function of the acetyl-coA
transporter could be critical in allowing the reaction to pro-
ceed within the lumen of the Golgi apparatus. The marked
inhibition by palmitoyl-CoA suggests that it may almost be a
substrate for the translocation process. This could explain
how this molecule becomes available for the addition of pal-
mitate residues to proteins, which is known to take place in
the Golgi apparatus (52).
Previous studies with bovine submaxillary glands have been
interpreted to indicate that the 0-acetylation reaction can
take place either after the transfer of sialic acids to glycocon-
jugates or on free sialic acids in the cytosol (13-15). Our study
does not provide evidence to rule out the latter possibility,
but demonstrates the mechanism by which the donor molecule
can be translocated into and concentrated in the Golgi appa-
ratus for use by 0-acetyltransferases. Since CMP-sialic acid
is promptly used or hydrolyzed upon entry into the Golgi
apparatus (17), it is very unlikely that it could serve as an
intermediate substrate for the 0-acetyltransferases in the
Golgi lumen. It is most likely that the acetyl group is directly
donated to specific sialic acids of acceptor glycoconjugates in
transit through the Golgi apparatus. Recent studies have
shown that the Golgi apparatus is highly organized, with the
various activities involving glycoprotein biosynthesis being
appropriately distributed from the cis to the trans face (52-
54). If the acetyl-coA transport process is exclusively in-
tended for the 0-acetylation of sialic acids, one would predict
that it would be localized in the trans-most stacks, where the
sialylation reactions take place.
We have provided evidence that the entire acetyl-coA
molecule is involved in the translocation process. However,
the intravesicular low molecular weight fraction was found to
consist almost exclusively of free acetate. We have two pos-
sible explanations for this finding. First, we have found pre-
liminary evidence for an 0-acetyl-esterase within the Golgi
lumen that appears to be distinct from a recently described
cytosolic sialic acid 0-acetylesterase (55). Second, our data
suggest the existence of an acetyl-coA hydrolase in the Golgi
vesicles. We are not sure of the relationship of this activity
to previously described acetyl-coA hydrolases (56-58). Either
or both of these activities could be responsible for the gener-
6608
Acetyl-coA Uptake by Golgi Vesicles
ation of the free acetate found within the vesicles. We have
not followed the fate of the CoA released upon utilization
and/or hydrolysis of the acetyl-coA within the Golgi lumen.
Since CoA is known to be an inhibitor of the O-acetyltrans-
ferase(s), it would be important to eliminate it from this
location in an efficient manner. It is possible that an antiport
system akin to that recently described for the sugar nucleo-
tides
(23)
may be involved in acetyl-coA uptake and removal
of
GOA.
We are currently exploring this possibility.
Thus, it appears that the 0-acetylation of sialic acids in the
Golgi apparatus could be regulated by many different proc-
esses, including an acetyl-coA transporter, one or more sialic
acid-specific 0-acetyltransferases, an acetyl-coA hydrolase,
and an 0-acetylesterase. In addition, some recent data suggest
that there are substrate specificity factors involving the ac-
ceptor glycoconjugate itself. For example, of several ganglio-
sides in human melanoma cells, only two were selectively
0-
acetylated
(59,
60),
and in a mouse brain ganglioside, only
1
of
3
sialic acid residues was found to be 0-acetylated
(61).
The existence of such exquisitely tissue- and acceptor-specific
0-acetylation reactions suggests that they must have some
important biological roles.
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