Enzymatic formation of dehydrodolichal and dolichal, new products related to yeast dolichol biosynthesis.

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 x g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol. In double-labeled experiments with a combination of -l-14C-isopentenyl diphosphate and a [4B-3H]NADPH-generating system, the ratio of 3H- and 14C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109-10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.

Two new polyprenyl products in addition to dehydrodolichol and dolichol were detected by two-plate silica gel thin layer chromatography of nonpolar products formed from [1-14 C]isopentenyl diphosphate and farnesyl diphosphate in the reaction with a crude 1,000 ؋ g supernatant of yeast homogenates in the presence of NADPH. The new products were indistinguishable from authentic dehydrodolichal and dolichal. Analyses of the time-dependent and pH-dependent formation of the four products including dehydrodolichal and dolichal suggested that the biosynthetic pathway from dehydrodolichol leading to dolichal is different from that to dolichol.

In double-labeled experiments with a combination of [1-14 C]isopentenyl diphosphate and a [4B-3 H]NADPHgenerating system, the ratio of 3 H-and 14 C-derived radioactivities found in dolichal was six times higher than that in dolichol. A small amount of 3 H-labeled dehydrodolichol was also detected. Considering the fact that dolichol is synthesized from dehydrodolichol (Sagami, H., Kurisaki, A., and Ogura, K. (1993) J. Biol. Chem. 268, 10109 -10113), we propose that dehydrodolichol is a common branch point intermediate in the biosynthetic pathways leading to dolichal and dolichol and that dehydrodolichal is an intermediate in the pathway from dehydrodolichol to dolichal.
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 2 are identical with those of the biosynthesis of cholesterol, ubiquinone, and farnesylated proteins. The next step begins with the cis-condensation of isopentenyl-P 2 with farnesyl-P 2 to form dehydrodolichyl-P 2 . It has been proposed that in the chain elongation reactions are involved two steps comprising farnesyl-P 2 3 Z,E,E-geranylgeranyl-P 2 and Z,E,E-geranylgeranyl-P 2 3 dehydrodolichyl-P 2 (10,11). The terminal biosynthetic pathway still remains to be clarified, although four mechanisms have been proposed (12,13): 1) dehydrodolichyl-P 2 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 isopen-tenol instead of isopentenyl-P 2 is added is accompanied by concomitant reduction to yield dolichol; and 4) dehydrodolichol produced by dephosphorylation of dehydrodolichyl-P 2 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 temperaturesensitive 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 2 -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). [1-14 C]Isopentenyl-P 2 (52 Ci/mol) and [1-3 H]glucose (15.0 Ci/mmol) were purchased from Amersham Corp. and American Radiolabeled Chemicals, respectively. Farnesyl-P 2 was prepared according to the method of Davisson et al. (14). Dolichol with major chain lengths of C 90 and C 95 , dehydrodolichol with major chain lengths of C 85 and C 90 , 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 reversephase 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 2 (300 mg) in 3 ml of chloroform at room temperature for 24 h. The reaction mixture was centrifuged at 10,000 ϫ 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 ϫ 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 ϫ 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 [1-14 C]isopentenyl-P 2 , 40 mM farnesyl-P 2 , 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) 1 were prepared by a sonication method similar to that of Van Dessel and Lagrou (19). Phosphatidylcholine (3 mmol) and [1-14 C]dehydrodolichol (0.18 mCi) were mixed in chloroform:methanol (1:1, v/v). The mixture was evaporated under N 2 , 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 3 . The suspension was then sonicated for 6 min (Branson Sonifier 200) on ice and centrifuged at 20,000 ϫ g for 20 min. Quantitative encapsulation of [1-14 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 ϫ 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-3 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 [1-3 H]glucose (1 mCi), 1 mM ATP, 10 mM MgCl 2 , 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 2ϩ . [1-3 H]Glucose-6-phosphate in the run-through fraction was incubated with 0.5 mmol of NADP ϩ 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.

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 ϫ g supernatant of yeast homogenates. The activity for dolichol synthesis was the highest in the latelogarithmic phase of growth. Therefore, in the following exper-iments we harvested the cells before the stationary phase (A 600 nm , 1.0). Next we prepared 1,000, 3,000, 5,000, 10,000, and 100,000 ϫ g supernatants of homogenates and assayed their activities for synthesis of dehydrodolichyl compounds and dolichol (Fig. 1B). The 1,000 ϫ g supernatant had an activity for dolichol synthesis about 10 times as high as that of the 10,000 ϫ g supernatant. The 100,000 ϫ 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 ϫ 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 ϫ 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 [1-14 C]isopentenyl-P 2 and farnesyl-P 2 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).
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 1 The abbreviations used are: SUV, small unilamellar vesicle; CHAPS, source of dolichyl-P (21). We prepared a 1,000 ϫ g supernatant of each mutant pretreated at the permissive temperature (23°C) or the restrictive temperature (37°C), incubated the supernatant with [1-14 C]isopentenyl-P 2 and farnesyl-P 2 , 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 temperaturesensitive 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-3 H]NADPH and [4A-3 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 ϫ g supernatant showed that NADP ϩ 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 ϩ . Extended dialysis of the 1,000 ϫ g supernatant considerably reduced the effects of the endogenous system on dolichol formation. Fig. 3 shows the effects of the concentration of glucose 6-phosphate or NADP ϩ on the formations of dolichol and squalene in NADPH-gener-ating 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 ϩ or with 0.5 mM NADP ϩ in the presence of 1 mM glucose 6-phosphate. The formation of dolichol (Fig. 3A) was increased with increasing concentrations of NADP ϩ 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 ϩ 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-3 H]NADPH formed from NADP ϩ and [1-3 H]glucose-6phosphate 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 ϩ /NADPH-dependent dehydrogenases.
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 2 . 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 4 or LiAlH 4 . 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 2 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 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.
Effect of NADP ϩ and NADPH on the Formation of Dehydrodolichol, Dehydrodolichal, Dolichal, and Dolichol-We examined the time-dependent formation of dehydrodolichol, dehy-drodolichal, dolichal, dolichol, and squalene in the in vitro system (Table I). In the case of assays with NADP ϩ , 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 ϩ 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 ϩ and NADPH-Since NADP ϩ /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 ϩ and NADPH (Table II). 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 [1-14 C] isopentenyl-P 2 as a single precursor in the presence of the [4B-3 H]NADPH-generating system. Table III shows the radioactivity of 3 H and 14 C in dehydrodolichol, dehydrodolichal, dolichal, dolichol, and squalene fractions on the two-plate thin layer chromatogram. All the detectable compounds contained 3 H-derived radioactivity. Assuming that one hydride from NADPH is incorporated into one molecule of dolichal or dolichol, the ratio of 3 H-to 14 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 4 . 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 reversephase C 18 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 local-FIG. 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 ϫ 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 70 -C 90 , C 75 -C 80 , C 65 -C 95 , and C 70 -C 90 , respectively with the major one being C 80 . ized 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 2 and farnesyl-P 2 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 2 and farnesyl-P 2 , 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 2 and farnesyl-P 2 . 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 2 -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 2 -dolichol.
To elucidate the stereochemistry of the hydride transfer from NADPH in dolichol biosynthesis, we prepared [4B-3 H]NADPH and [4A-3 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 ϩ , 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 synthe-sized directly from dehydrodolichol by the action of NADPHdependent reductase. If NADP ϩ and NADPH are both necessary to form dolichol from dehydrodolichol, it could be postulated that dehydrodolichol is first oxidized to dehydrodolichal by an NADP ϩ -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 3 H-derived radioactivity from [4B-3 H]NADPH. To our surprise, the dehydrodolichol fraction also contained a small but significant amount of 3 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 3 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 4Bhydride of NADPH is incorporated into one molecule of

TABLE III Assays with [4B-3 H]NADPH-generating system
The enzymatic assays were conducted as described under "Experimental Procedures" except that treatment with acid phosphatase was omitted and that the incubation was carried out overnight. A 1,000 ϫ g supernatant of mutant no. 149 was used for the assay. The theoretical ratios of 3 Hto 14 C-radioactivities were calculated on the basis of the assumption that the number of average isoprene units constituting the carbon chain of dolichal or dolichol are 16. Dedolichol, dehydrodolichol; dedolichal, dehydrodolichal. 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 ϩ to an assay mixture containing NADPH enhanced the activity of dolichol synthesis. 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.