Since the discovery of the involvement of the long-chain
polyprenyl-P, dolichyl-P, in the pathway of assembly of the
oligosaccharide chain of N-linked
glycoproteins(1, 2) , a great deal of progress has
been made in the understanding of the biosynthetic pathway of
dolichol(3, 4, 5, 6, 7, 8, 9) .
The initial steps from mevalonate to farnesyl-P
are
identical with those of the biosynthesis of cholesterol, ubiquinone,
and farnesylated proteins. The next step begins with the cis-condensation of isopentenyl-P
with
farnesyl-P
to form dehydrodolichyl-P
. It has
been proposed that in the chain elongation reactions are involved two
steps comprising farnesyl-P
Z,E,E-geranylgeranyl-P
and Z,E,E-geranylgeranyl-P
dehydrodolichyl-P
(10, 11) . The terminal
biosynthetic pathway still remains to be clarified, although four
mechanisms have been proposed(12, 13) : 1)
dehydrodolichyl-P
is converted into dolichyl-P by
dephosphorylation followed by saturation of the
-isoprene
double-bond; 2) dolichol is the end product from which dolichyl-P is
produced by phosphorylation; 3) the final condensation in which
isopentenol instead of isopentenyl-P
is added is
accompanied by concomitant reduction to yield dolichol; and 4)
dehydrodolichol produced by dephosphorylation of
dehydrodolichyl-P
is reduced to dolichol.
We have
demonstrated that the fourth mechanism is operative for the dolichol
biosynthetic pathway, since exogenous dehydrodolichol was shown to be
converted to dolichol under in vitro assay conditions with
preparation from mammalian liver and testis(13) . However, the
conversion of dehydrodolichol into dolichol was too low to study on the
mechanisms. Recently, Roos et al.(21) described a
novel screen for yeast temperature-sensitive mutants with defects in
the dolichol-mediated pathway for N-glycosylation of proteins.
In their experiments the mutants were further classified into phase 1,
2, and 3 mutants with defects in the pathways for the formation of
dolichyl-P, oligosaccharyl-P
-dolichol, and N-linked oligosaccharyl proteins, respectively. In order to
understand the terminal biosynthetic pathway to dolichol, we planned to
establish an in vitro dolichol assay method for yeast enzyme
systems and to search among the phase 1 mutants for one which might be
a mutant defective in the reduction of dehydrodolichol. We also were
interested in learning if the hydride of NADPH is incorporated into
dolichol and, if so, if the 4A- or the 4B-hydride of NADPH is
stereospecifically incorporated. In the course of these studies, we
have found two new enzymatic products, namely dehydrodolichal and
dolichal. These results are discussed in the context of the terminal
step in dolichol biosynthesis.
EXPERIMENTAL PROCEDURES
Materials
Saccharomyces cerevisiae haploid strain A364A (MAT
ade1 ade2 ura1
lys2 tyr1 his7 gal1) used as wild type and all mutants were
described earlier (21) .
[1-
C]Isopentenyl-P
(52 Ci/mol) and
[1-
H]glucose (15.0 Ci/mmol) were purchased from
Amersham Corp. and American Radiolabeled Chemicals, respectively.
Farnesyl-P
was prepared according to the method of Davisson et al.(14) . Dolichol with major chain lengths of
C
and C
, dehydrodolichol with major chain
lengths of C
and C
, ethyl geranylgeranoate,
and ethyl geranylneroate were generous gifts from Kuraray Co.
-Dihydrohexaprenol was kindly provided by Eizai Co. Acid
phosphatase (type II, from potato), hexokinase (type C-300, from
bakers' yeast), glucose-6-phosphate dehydrogenase (type VII, from
bakers' yeast), and Schiff's reagent were purchased from
Sigma. Silica Gel 60 thin layer and reverse-phase LKC-18 thin layer
plates were purchased from Merck and Whatman, respectively. RP-18
Sep-Pak and Dowex 50W-X8 were purchased from Waters and The Dow
Chemical Company, respectively. All other chemicals were of reagent
grade.
Synthesis of Dehydrodolichal and
Dolichal
Dehydrodolichal was prepared from dehydrodolichol
according to the method of Attenburrow et al.(15) .
Dehydrodolichol (30 mg) was treated with active MnO
(300
mg) in 3 ml of chloroform at room temperature for 24 h. The reaction
mixture was centrifuged at 10,000
g for 10 min to
remove brown pellets. The chloroform extracts containing
dehydrodolichal were dried under vacuum, dissolved in toluene, and
purified by silica gel column chromatography in toluene. In the case of
dolichal synthesis, dolichol (10 mg) was treated with pyridinium
dichromate in 0.35 ml of dichloromethane at room temperature for 24 h
according to the method of Corey and Schmidt (16) . The
reaction mixture was centrifuged at 10,000
g for 10
min to remove brown pellets. The dichloromethane extracts containing
dolichal were dried under vacuum, dissolved in toluene, and purified by
similar chromatography to that of dehydrodolichal in toluene. Both
aldehydes were quantitatively obtained.
Enzyme Preparations and Assay
Yeast cells were
grown in YPD medium (2% Bacto-peptone, 1% Bacto-yeast extract, 2%
glucose, 0.003% adenine sulfate) at 23 °C to late logarithmic
phase, collected, washed, suspended in 100 mM Tris-HCl (pH
7.5) containing 1 mM dithiothreitol, and incubated at 23
°C for 5 min. The cells were collected, resuspended in a buffer
containing 10 mM Tris-HCl (pH 7.5), 1.2 M sorbitol,
0.75% Bacto-yeast extract, 1.5% Bacto-peptone, 0.5% glucose, and 0.2
mg/ml Zymolyase 100T, and then incubated at 23 °C for 30 min.
Spheroplasted cells were collected, washed with 1.2 M sorbitol, resuspended in a preparation buffer containing 20 mM Hepes-KOH (pH 7.5), 1 mM potassium acetate, 2 mM magnesium acetate, 1 mM dithiothreitol, 1 mM EDTA, 1 mM EGTA, 0.6 mg/ml protease inhibitors
(leupeptin, antipain, chymostatin, pepstatin A, aprotinin), and 1
mM phenylmethylsulfonyl fluoride, and homogenized with 30
strokes in a Teflon glass homogenizer. The homogenates were centrifuged
at 1,000
g for 30 min, and the resulting supernatant
was used as crude enzyme. Protein was determined with DC protein assay
reagents (Bio-Rad). Mutant crude enzymes were prepared as described
above except that cells were grown at 23 °C to mid-logarithmic
phase and then divided in two equal aliquots; one aliquot was incubated
at 36 °C for 90 min and the other remained at 23 °C.
Assay Conditions and Products Analysis
The
standard assay mixture contained, in a final volume of 0.5 ml, Tris-HCl
buffer (pH 8.0), 1 mM dithiothreitol, 100 mM patassium fluoride, 50 mM [1-
C]isopentenyl-P
, 40 mM farnesyl-P
, 1 mM NADPH, and 2.5 mg/ml crude
enzymes. The mixture was incubated at 23 °C for 30 min. The
enzymatic products were extracted with butanol saturated with water.
The butanol extracts were treated with acid phosphatase according to
the method of Fujii et al.(17) . Liberated products
were extracted with hexane, and the hexane extracts were passed through
RP-18 Sep-Pak in methanol. Nonpolar products were eluted with hexane
and analyzed by two-plate thin layer chromatography as described in a
previous report(18) .
Preparation of Phospholipid Vesicles
Small
unilamellar vesicles (SUVs) (
)were prepared by a sonication
method similar to that of Van Dessel and Lagrou(19) .
Phosphatidylcholine (3 mmol) and
[1-
C]dehydrodolichol (0.18 mCi) were mixed in
chloroform:methanol (1:1, v/v). The mixture was evaporated under
N
, dried under vacuum for 3 h, and then suspended in 1 ml
of a preparation buffer containing 50 mM Tris-HCl (pH 8.0),
0.25 M sucrose, 1 mM EDTA, and 0.02% NaN
.
The suspension was then sonicated for 6 min (Branson Sonifier 200) on
ice and centrifuged at 20,000
g for 20 min.
Quantitative encapsulation of
[1-
C]dehydrodolichol in SUVs was confirmed by
liquid scintillation counting of an aliquot (50 ml) of the supernatant.
The supernatant (650 ml) was used as SUVs source. The liposome and
1,000
g supernatant of yeast homogenates were
incubated as described under ``Assay Conditions and Products
Analysis,'' except that the assay mixture contained 0.25 M sucrose.
Double-labeled Experiments
The application of
[4B-
H]NADPH generating system in double-labeled
experiments was performed by the modified method of Moran et
al.(20) . A mixture containing, in a final volume of 0.2
ml, 50 mM Tris-HCl buffer (pH 7.8), 0.33 mM [1-
H]glucose (1 mCi), 1 mM ATP, 10
mM MgCl
, and 0.23 unit of hexokinase was incubated
at 25 °C for 2 h. The reaction products were passed through a Dowex
50W-X8 column (0.5 ml) in water to remove Mg
.
[1-
H]Glucose-6-phosphate in the run-through
fraction was incubated with 0.5 mmol of NADP
and 0.5
unit of glucose-6-phosphate dehydrogenase, in a total volume of 0.3 ml,
at 23 °C for 5 min. Then, the preincubated mixture was added to the
standard assay mixture.
RESULTS
Establishment of the in Vitro Dolichol Assay in
Yeast
To confirm whether the formation of dolichol by yeast
enzyme preparations can be detected, we first applied the assay system
established with crude enzyme preparations of rat liver to the yeast
system. Fig. 1A shows growth of yeast and synthesis of
dolichol with a 10,000
g supernatant of yeast
homogenates. The activity for dolichol synthesis was the highest in the
late-logarithmic phase of growth. Therefore, in the following
experiments we harvested the cells before the stationary phase (A
, 1.0). Next we prepared 1,000, 3,000,
5,000, 10,000, and 100,000
g supernatants of
homogenates and assayed their activities for synthesis of
dehydrodolichyl compounds and dolichol (Fig. 1B). The
1,000
g supernatant had an activity for dolichol
synthesis about 10 times as high as that of the 10,000
g supernatant. The 100,000
g supernatant had no
activity for dolichol synthesis, although it had a considerable
activity for the synthesis of dehydrodolichyl compounds. Therefore, we
used the 1,000
g supernatant to examine the assay
conditions concerning optimum pH, time, and NADPH dependence. We found
that there was no remarkable improvement of the dolichol assay
conditions earlier established for the 10,000
g supernatant of rat liver homogenates(13) . We also
examined the effects of various detergents such as Triton X-100, CHAPS,
octyl glucopyranoside, deoxycholate, and Tween 80 on the activity of
dolichol synthesis. As shown in Fig. 2, all the detergents
except Tween 80 completely at their critical micellar concentrations
inhibited the activity of dolichol synthesis, whereas the activity for
dehydrodolichol synthesis was less affected. Since the specific
activity of the yeast enzyme system was higher than that of the rat
liver enzyme system, in these studies a protein concentration of 2.5
mg/ml was used in the following experiments. Analysis of products
derived from [1-
C]isopentenyl-P
and
farnesyl-P
with or without acid phosphatase treatment
revealed the absence of dolichyl diphosphate and dolichyl phosphate in
the enzymatic products and no difference in the chain length
distribution between dehydrodolichyl products and dolichol (data not
shown).
Figure 1:
Activities for dolichol
synthesis in yeast. A, growth of yeast and synthesis of
dolichol (bars). The figures on the top of the bars indicate
the radioactivity of dolichol synthesized. B, formation of
dehydrodolichyl products (left panel) and dolichol (right
panel) in the reaction with the supernatant fraction resulting
from differential centrifugation of yeast homogenates. The enzymatic
assays were conducted as described under ``Experimental
Procedures.''
Figure 2:
Effects of the concentration of several
detergents on the formation of dehydrodolichyl products (
) and
dolichol (
). The enzymatic assays were conducted as described
under ``Experimental Procedures.'' A, Triton X-100; B, CHAPS; C, octylglucopyranoside; D,
deoxycholate; E, Tween 80.
Dolichol-synthesizing Activities of Temperature-sensitive
Yeast Mutants
The mutant strain nos. 64, 149, 283, 279, and 358
used in this study are phase 1 mutants thought to be defective in the
biosynthesis reactions responsible for the source of
dolichyl-P(21) . We prepared a 1,000
g supernatant of each mutant pretreated at the permissive
temperature (23 °C) or the restrictive temperature (37 °C),
incubated the supernatant with
[1-
C]isopentenyl-P
and
farnesyl-P
, and analyzed the products by two-plate thin
layer chromatography. Dolichol was formed in all of these mutants
pretreated at the restrictive temperature. A mutant (no. 283), which
has been already identified to be allelic to sec 59, which is a
temperature-sensitive mutant defective in dolichol kinase activity, was
almost equal to the wild type strain in the capability of synthesizing
dolichol. However, surprisingly, the abilities to synthesize
dehydrodolichyl compounds and dolichol by the mutant nos. 64, 149, 279,
and 358 were higher than that of the wild type strain. Remarkably,
these mutants seem to have a mutation which causes an increase in in vitro activity involved in the formation of dehydrodolichyl
compounds and dolichol. We chose to use the mutant, #149 in the
following experiments.
Enzymatic Synthesis of Dehydrodolichal from
Dehydrodolichol
To see whether the hydride of NADPH is in fact
incorporated into dolichol and whether the hydride is derived from 4B-
or 4A-hydrogen of NADPH, we tried to prepare stereospecifically labeled
[4B-
H]NADPH and [4A-
H]NADPH
according to the method of Moran et al.(20) . However,
we had difficulty in preparing and purifying labeled NADPH in amounts
enough for the minimum concentration of 0.1 mM in the standard
assay mixture for dolichol formation. We took another approach using an
NADPH-generating system to assay the incorporation of the hydrogen of
NADPH into dolichol, because this approach was expected to provide in situ even such high concentrations of labeled NADPH as 1.0
mM. Preliminary experiments with a crude 1,000
g supernatant showed that NADP
rather than NADPH
had a stimulatory effect on the activity for dolichol synthesis,
suggesting the occurrence of an endogenous system of synthesizing NADPH
from NADP
. Extended dialysis of the 1,000
g supernatant considerably reduced the effects of the
endogenous system on dolichol formation. Fig. 3shows the
effects of the concentration of glucose 6-phosphate or NADP
on the formations of dolichol and squalene in NADPH-generating
systems. In the case of squalene (Fig. 3B) synthesis,
the formation was saturated with the addition of 0.1 mM glucose 6-phosphate in the presence of 1 mM NADP
or with 0.5 mM NADP
in the presence of 1 mM glucose 6-phosphate. The
formation of dolichol (Fig. 3A) was increased with
increasing concentrations of NADP
in the presence of 1
mM glucose 6-phosphate, whereas it was saturated at a
concentration of 0.1 mM glucose 6-phosphate in the presence of
1 mM NADP
and then decreased with the
increase of the concentration of glucose 6-phosphate. The amount of
formation of dolichol with the NADPH-generating system was larger than
that with 1 mM NADPH alone. We next examined the incorporation
of tritium of [4B-
H]NADPH formed from
NADP
and
[1-
H]glucose-6-phosphate by the NADPH-generating
system into dolichol. Significant incorporation of radioactivity into
the dolichol fraction (903 dpm) was observed. To our surprise, the
dehydrodolichol fraction also contained radioactivity (1,345 dpm). This
implies that dehydrodolichol and dehydrodolichal are interconvertible
by the action of NADP
/NADPH-dependent dehydrogenases.
Figure 3:
Effects of the NADPH-generating system on
dolichol and squalene syntheses. Effects of the concentration of
glucose 6-phosphate (gray bars) in the presence of 1 mM NADP
and the concentration of NADP
(hatched bars) in the presence of 1 mM glucose
6-phosphate on the formation of dolichol (A) and squalene (B) were examined by use of the NADPH-generating system. The black bars correspond to the values when the NADPH-generating
system was replaced with 1 mM NADPH. The enzymatic assays were
conducted as described under ``Experimental Procedures''
except that treatment with acid phosphatase was omitted and that
overnight incubation was carried out. A 1,000
g supernatant of mutant no. 149 was used for the
assay.
Chemical Synthesis of Dehydrodolichal and
Dolichal
To see the chemical behavior of dehydrodolichal on
silica gel thin layer chromatography, we chemically synthesized an
authentic specimen of dehydrodolichal by treating dehydrodolichol with
MnO
. We also synthesized dolichal by treatment of dolichol
with pyridinium dichromate for comparison. As shown in Fig. 4A, the dehydrodolichal compound (lane 2)
migrated much slower than the dolichal compound (lane 6) on a
silica gel thin layer plate, although dehydrodolichol (lane 1)
had a little greater mobility than dolichol (lane 5). To
confirm that these compounds are aldehydes, they were treated with
NaBH
or LiAlH
. Both compounds were reduced to
dehydrodolichol (lanes 3 and 4) and dolichol (lanes 7 and 8). These authentic compounds on a
silica gel plate also turned blue-violet on addition of a
Schiff's reagent, indicating that the two compounds obtained by
the treatment with MnO
and pyridinium dichromate are in
fact dehydrodolichal and dolichal, respectively. To see their
separation dependent on the carbon chain length, we compared these
aldehydes with each other on a reverse-phase silica gel plate (Fig. 4B). Dehydrodolichol (lane 3) migrated
with almost the same R value as dehydrodolichal (lane
4). A similar observation was made with dolichol (lane 5)
and dolichal (lane 6). When dehydrodolichol and its
corresponding dolichol are compared, dehydrodolichol (lane 1)
moved faster than dolichol (lane 2). A similar relation was
also observed in the case of dehydrodolichal (lane 7) and
dolichal (lane 8).
Figure 4:
Normal-phase (A) and
reverse-phase C
(B) silica gel thin layer
chromatograms of dehydrodolichyl and dolichyl compounds. A, lane 1, dehydrodolichol with the major chain length of
C
and C
; lane 2, dehydrodolichal
obtained by MnO
treatment of dehydrodolichol; lane
3, dehydrodolichol obtained by NaBH
treatment of
dehydrodolichal corresponding to lane 2; lane 4,
dehydrodolichol obtained by LiAlH
treatment of
dehydrodolichal corresponding to lane 2; lane 5,
dolichol with the major chain length of C
and
C
; lane 6, dolichal obtained by pyridinium
dichromate treatment of dolichol; lane 7, dolichol obtained by
NaBH4 treatment of dolichal corresponding to lane 6; lane
8, dolichol obtained by LiAlH
treatment of dolichal
corresponding to lane 6. B, lane 1 and 3,
dehydrodolichol with major chain length of C
and
C
; lanes 2 and 5, dolichol with the
major chain length of C
and C
; lanes 4 and 7, dolichal with the major chain length of C
and C
; lanes 6 and 8,
dehydrodolichal with the major chain length of C
and
C
.
Two-plate Chromatography
In order to search for
dehydrodolichal, which is expected to occur in enzymatic products, the
portion of the first silica gel plate on which radioactive nonpolar
products had been developed was subjected to the second reverse-phase
chromatography. As shown in Fig. 5, two new polyprenyl families
indicated by arrows 2 and 3 were observed in addition
to dehydrodolichol and dolichol families (arrows 4 and 5), respectively. The faint radioactivity family spots (arrow 3) were in good agreement to those of the authentic
dehydrodolichal family. Further, the radioactivity family spots (arrow 2) comigrated with authentic dolichal. Their migrations
were also confirmed by another two-plate silica gel chromatography
using a different solvent system, toluene:ethyl acetate (9:1). These
results indicate that not only dehydrodolichal but also dolichal is
formed in considerable amounts in these in vitro experiments.
Figure 5:
A two-plate thin layer radiochromatogram
of nonpolar products. The enzymatic assays were conducted as described
under ``Experimental Procedure'' except that treatment with
acid phosphatase was omitted and that the incubation was carried out
overnight. A 1,000
g supernatant of mutant no. 149 was
used for the assay. The first silica gel and the second reverse-phase
silica gel chromatography steps were carried out in solvent systems of
toluene:ethyl acetate (19:1) and acetone:methanol (9:1), respectively. Arrows 1, 2, 3, 4, and 5 point to squalene, dolichal, dehydrodolichal, dehydrodolichol, and
dolichol, respectively. The chain lengths of dolichal, dehydrodolichal,
dehydrodolichol, and dolichol were analyzed and found to be
C
-C
,
C
-C
,
C
-C
, and
C
-C
, respectively with the major one
being C
.
Effect of NADP
and NADPH on the
Formation of Dehydrodolichol, Dehydrodolichal, Dolichal, and
Dolichol
We examined the time-dependent formation of
dehydrodolichol, dehydrodolichal, dolichal, dolichol, and squalene in
the in vitro system (Table 1). In the case of assays
with NADP
, the formation of dehydrodolichal and
dolichal preceded that of dolichol. It is suggested that the formation
of squalene as well as dolichol is dependent on NADPH synthesized from
exogenous NADP
by the action of an endogenous
NADPH-generating system, although dialyzed enzyme preparations were
used. In the case of assays with NADPH, dolichal and dolichol were
predominantly synthesized over dehydrodolichal.
Effect of pH on the Formation of Dehydrodolichol,
Dehydrodolichal, Dolichal, and Dolichol in the Presence of
NADP
and NADPH
Since
NADP
/NADPH-dependent oxidoreduction seemed to be
involved in the biosynthetic pathway from dehydrodolichol to
dehydrodolichal, dolichal, and dolichol, we examined the effects of pH
on the formation of these products and squalene in the presence of both
NADP
and NADPH (Table 2). While the synthesis of
dolichol was optimal at pH 7-8, that of dolichal was optimal at
pH 8-9.
Incorporation of the 4B-Hydride of NADPH into
Dehydrodolichol, Dehydrodolichal, Dolichal, Dolichol, and
Squalene
To establish whether dolichol is formed from
dehydrodolichol directly or via intermediates such as dehydrodolichal
and dolichal, we performed double-labeled experiments with
[1-
C]isopentenyl-P
as a single
precursor in the presence of the
[4B-
H]NADPH-generating system. Table 3shows the radioactivity of
H and
C in dehydrodolichol, dehydrodolichal, dolichal, dolichol,
and squalene fractions on the two-plate thin layer chromatogram. All
the detectable compounds contained
H-derived radioactivity.
Assuming that one hydride from NADPH is incorporated into one molecule
of dolichal or dolichol, the ratio of
H- to
C-derived radioactivity is calculated to be 18 for each
compound. Based on this value, the specific activities of NADPH
utilized in the syntheses of dolichal and dolichol were estimated to be
about 2- and 12-fold diluted, respectively. The dilution of NADPH in
the case of dolichol synthesis was in good agreement to that in the
case of squalene synthesis. These results indicate that the site for
biosynthesis of dolichal is different from that of dolichol and that
there is no direct interconversion between dolichal and dolichol.
Further Product Analyses
To determine whether
enzyme preparations of a wild type yeast as well as mutant no. 149 have
also the ability to synthesize dehydrodolichal and dolichal in addition
to dehydrodolichol and dolichol, we analyzed the enzymatic products.
Dolichal, dehydrodolichol, and dolichol were formed. The formation of
dehydrodolichal was too small to detect. The radioactive dolichal
fraction was converted 89% to dolichol by treatment of
LiAlH
. No chemical oxidation of dehydrodolichol or dolichol
into dehydrodolichal or dolichal was observed in experiments (23 or 50
°C, pH 9 or 10, overnight incubation). Further, we also analyzed
naturally occurring nonpolar polyprenyl compounds of a wild type yeast.
Dolichol was easily detected, but dehydrodolichol, dehydrodolichal, or
dolichal were not detected. We have found unknown compounds which
migrated faster than dolichal on normal-phase silica gel thin layer
chromatography and which were separated into a family with respect to
carbon chain length on reverse-phase C
silica gel thin
layer chromatography. This compound was resistant to mild alkaline
hydrolysis.
DISCUSSION
We have previously demonstrated the formation of dolichol
from dehydrodolichol by a NADPH-dependent reductase localized in
microsomes of mammalian liver and testis(13) . However, the
reductase activity for dehydrodolichol was extremely low, and even in
an assay using a combination of isopentenyl-P
and
farnesyl-P
did not yield sufficient amounts of dolichol for
further studies. We therefore employed yeast cells instead of mammalian
tissue and found that yeast enzyme preparations had a much higher
specific activity than mammalian enzyme preparations. More than 10 mg
of protein/ml were required in the assay to detect the mammalian
activity for dolichol synthesis from isopentenyl-P
and
farnesyl-P
, whereas 2.5 mg of protein/ml were sufficient
for the detection of the same activity in the case of yeast enzyme.
We tried to examine whether the yeast enzyme activity was high
enough to utilize exogenous dehydrodolichol as a precursor for the
reductase. However, dehydrodolichol was not effectively utilized as a
substrate in the yeast system, even under conditions where the compound
was solubilized with Tween 80 and unilamellar phospholipid vesicles.
Because of the difficulty in employing dehydrodolichol as a substrate
for the yeast enzymes, we decided to improve the assay method by
replacing the alcohol substrate with a combination of
isopentenyl-P
and farnesyl-P
. We first applied
this assay to measure the enzyme activities of several yeast mutants
that have been identified to be defective in the accumulation of
oligosaccharyl-P
-dolichol. We were particularly interested
in the possibility that one or more of these mutants might contain a
temperature sensitive enzyme involved in the reduction of
dehydrodolichol. However, we could not detect temperature-sensitivity
as far as the dolichol biosynthetic pathway was concerned. Instead, we
made the surprising finding that all the mutants except mutant no. 283
had much higher activities for dolichol synthesis than the wild type
strain. The mechanism of this change is not known. Perhaps the
induction of enzymes responsible for dolichol biosynthesis might result
from compensation for the reduced levels of dolichyl-P or
oligosaccharyl-P
-dolichol.
To elucidate the
stereochemistry of the hydride transfer from NADPH in dolichol
biosynthesis, we prepared [4B-
H]NADPH and
[4A-
H]NADPH. However, the yields of both
radioactive forms of NADPH thus obtained were too low to satisfy even
the minimum concentration of NADPH established for the yeast enzyme
assay. Therefore, we employed an NADPH-generating system for
elucidation of the stereochemistry of NADPH reduction. The crude enzyme
preparation was dialyzed sufficiently to remove at least free glucose
6-phosphate, because it was found that dolichol was formed with crude
nondialyzed enzymes without addition of exogenous glucose 6-phosphate.
In the reaction with the dialyzed enzymes, the formation of dolichol
was increased with increasing concentration of exogenous glucose
6-phosphate. The activity with the NADPH-generating system containing
NADP
, glucose 6-phosphate, and glucose-6-phosphate
dehydrogenase was much higher than that with NADPH alone. These
puzzling results were readily reproduced. This observation raised the
possibility that dolichol is not synthesized directly from
dehydrodolichol by the action of NADPH-dependent reductase. If
NADP
and NADPH are both necessary to form dolichol
from dehydrodolichol, it could be postulated that dehydrodolichol is
first oxidized to dehydrodolichal by an NADP
-dependent
dehydogenase, and then the dehydrodolichal is reduced to dolichol by an
NADPH-dependent reductase. In a preliminary doubly-labeled experiment,
we found that the dolichol fraction contained
H-derived
radioactivity from [4B-
H]NADPH. To our surprise,
the dehydrodolichol fraction also contained a small but significant
amount of
H-derived radioactivity. This finding led us to
search for a new polyprenyl compound such as dehydrodolichal.
As
shown in Fig. 5, nonpolar products were separated into
dehydrodolichol, dehydrodolichal, dolichal, and dolichol in addition to
squalene. We assumed the biosynthetic pathway of these polyprenyl
products as follows, dehydrodolichol
dehydrodolichal
[arrow] dolichal [arrow] dolichol. The involvement
of these intermediates in the biosynthetic pathway to dolichol from
dehydrodolichol was attractive, because the hydride transfer onto the
-carbon of
,
-unsaturated carbonyl groups is well known.
However, experiments concerning time-dependent and pH-dependent
formation of these compounds revealed that these new products and
dolichol differed from each other in the biosynthetic pathway from
dehydrodolichol. Double-labeled experiments also showed a different
biosynthetic pathway to dolichol from to dolichal. On the basis that
one 4B-hydride of NADPH is incorporated into one molecule of squalene,
the numbers of 4B-hydride incorporated into dolichol and dolichal are
estimated to be one and six, respectively. However, it is difficult to
explain the large difference in hydride incorporation between dolichol
and dolichal. One possible reason is that the biosynthetic site for
dolichal formation might be more accessible to exogenous NADPH than
that for dolichol formation. Therefore, at present we propose that
dolichal is synthesized from dehydrodolichol via dehydrodolichal and
that dolichol is formed directly from dehydrodolichol (Fig. 6).
However, we do not exclude the possibility that dolichol is formed from
dehydrodolichal, because in a preliminary study the addition of
NADP
to an assay mixture containing NADPH enhanced the
activity of dolichol synthesis.
Figure 6:
Possible biosynthetic pathways of dolichol
and dolichal.
Steen et al.(22) have reported the identification of dolichyl
dolichoate in bovine thyroid. The formation of dehydrodolichal is well
understood as being involved in the biosynthesis of the dolichoate.
Thus, the dolichoate pathway might be different from the dolichol
pathway, which is responsible for the formation of dolichyl-P, a sugar
carrier lipid in N-linked glycoproteins and glycosyl
phosphatidyl inositol-anchored proteins.