©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Complementation of Saccharomycescerevisiae Strains Containing Fatty Acid Activation Gene ( FAA) Deletions with a Mammalian Acyl-CoA Synthetase (*)

Laura J. Knoll , D. Russell Johnson , Jeffrey I. Gordon (§)

From the (1) Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Four unlinked fatty acid activation ( FAA) genes encoding acyl-CoA synthetases have been identified in Saccharomyces cerevisiae and characterized by noting the phenotypes of isogenic strains containing all possible combinations of faa null alleles. None of these genes is required for vegetative growth when acyl-CoA production by the fatty acid synthetase (Fas) complex is active. When Fas is inhibited by cerulenin, exponentially growing cells are not viable on media containing a fermentable carbon source unless supplemented with fatty acids such as myristate, palmitate, or oleate. The functionally interchangeable FAA1 and FAA4 genes are responsible for activation of these imported fatty acids. Analysis of lysates prepared from isogenic FAA1FAA4 and faa1faa4 strains indicated that Faa1p and Faa4p together account for 99% of total cellular myristoyl-CoA and palmitoyl-CoA synthetase activities. Genetic complementation studies revealed that rat liver acyl-CoA synthetase (RLACS) rescues the viability of faa1faa4 cells in media containing a fermentable carbon source, myristate or palmitate, plus cerulenin. Rescue is greater at 37 °C compared with 24 °C, paralleling the temperature-dependent changes in RLACS activity in vitro as well as the enzyme's ability to direct incorporation of tritiated myristate and palmitate into cellular phospholipids in vivo. Complementation by RLACS is blocked by treatment of cells with triacsin C (1-hydroxy-3-( E, E, E,2`,4`,7`-undecatrienylidine)triazene). Even though Faa1p, Faa4p, and RLACS are all able to activate imported myristate and palmitate in S. cerevisiae, the sensitivity of Faa4p and RLACS, but not Faa1p, to inhibition by triacsin C suggests that the rat liver enzyme is functionally more analogous to Faa4p than to Faa1p. Finally, an assessment of myristate and palmitate import into FAA1FAA4 and faa1faa4 strains, with or without episomes that direct overexpression of Faa1p, Faa4p or RLACS, indicated that fatty acid uptake is not coupled to activation in S. cerevisiae.


INTRODUCTION

Regulation of myristoyl-CoA pool size in Saccharomyces cerevisiae has an important role in modulating the activity of myristoyl-CoA:protein N-myristoyltransferase, an enzyme essential for vegetative growth (Johnson et al., 1994a). There are at least two metabolic pathways in S. cerevisiae that produce myristoyl-CoA: de novo synthesis or activation of free myristate by acyl-CoA synthetases. The de novo pathway uses malonyl-CoA, produced by acetyl-CoA carboxylase ( Acc1p) (Mishina et al., 1980), to generate long chain saturated acyl-CoAs through the enzymatic activities contained within the cytosolic fatty acid synthetase (Fas)() complex (Paltauf et al., 1992). Palmitoyl-CoA and stearoyl-CoA are the principal products of Fas, while myristoyl-CoA represents 3-5% of the total acyl-CoAs synthesized (Hori et al., 1987). Cerulenin, a 12-carbon amide ((2 R,3 S)-2,3-epoxy-4-oxo-7,10- trans,trans-dodecanienamide; see Fig. 1), is a specific inhibitor of Fas (Nomura et al., 1972; Vance, 1972; Funabashi et al., 1989). When Fas is inhibited with cerulenin, wild-type strains of S. cerevisiae require supplementation of media with fatty acids such as myristate, palmitate, or oleate to remain viable. These fatty acids are imported and then activated by cellular acyl-CoA synthetases (Duronio et al., 1992; Johnson et al., 1994b, 1994c).


Figure 1: Comparison of the structures of cerulenin ((2 R,3 S)-2,3-epoxy-4-oxo-7,10- trans,trans-dodecanienamide), triacsin C (1-hydroxy-3-( E,E,E,2`,4`,7`-undecatrienylidine)triazene), and myristic acid (tetradecanoic acid). The overall length of cerulenin is equivalent to lauramide (CH(CH)CO-NH). The trans- or E-double bonds present at the 7,8 and 10,11 positions of cerulenin are also present in triacsin C: i.e. they share a pentadiene, -CH=CH-CH-CH=CH-. The alignment of cerulenin and triacsin C also reveals that polar groups are present at the right hand terminus of both molecules: there are three contiguous sp or sp-like (the epoxide carbon) atoms in cerulenin while triacsin C has an N-hydroxytriazene moiety essential for its inhibitory activity (Tomoda et al., 1987).



The overall reaction catalyzed by an acyl-CoA synthetase is as follows (Berg, 1956, 1958; Bar-Tana et al., 1973a, 1973b).

On-line formulae not verified for accuracy

S. cerevisiae contains at least five genes encoding acyl-CoA synthetases (or fatty acid activation proteins). Four unlinked FAA genes have been recovered to date (Duronio et al., 1992; Johnson et al., 1994b, 1994c). Faa1p, Faa2p, and Faa3p have been expressed in and purified from strains of Escherichia coli that do not produce FadD, the only acyl-CoA synthetase synthesized by this bacterium (Knoll and Gordon, 1993; Knoll et al., 1994). Faa1p and Faa2p can accommodate C7-C18 saturated fatty acids as substrates in vitro (optimum = C12:0-C16:0 for Faa1p and C9:0-C13:0 for Faa2p). Faa3p prefers C16 and C18 fatty acids with a cis-double bond at C9-C10 (Knoll et al., 1994).()

The functional contributions of the Faas to lipid metabolism have been examined by comparing the growth characteristics of isogenic strains with (i) NMT1 or a mutant allele ( nmt1-181) that causes temperature-sensitive growth arrest and myristic acid auxotrophy due to a reduction in the enzyme's affinity for myristoyl-CoA, (ii) various combinations of wild-type and null FAA alleles, and (iii) an active or cerulenin-inactivated de novo pathway for acyl-CoA synthesis (Johnson et al., 1994b, 1994c). The results indicate that if Fas is active and NMT1 cells are grown on rich media containing a fermentable carbon source ( e.g. dextrose), none of the FAA genes are needed for vegetative growth. However, when Fas is inactivated, maintenance of the viability of NMT1 cells requires that Faa1p, and to a lesser extent Faa4p, activate imported fatty acids (Johnson et al., 1994c). Faa2p appears to be unable to activate imported fatty acid substrates ( e.g. myristate or palmitate) but is involved in activation of endogenous fatty acids. None of the four FAAs are exclusively responsible for targeting imported fatty acids to perixosomal -oxidation pathways (Johnson et al., 1994c). Moreover, a NMT1 strain with deletions of all four FAA genes is still viable at 30 °C on media containing myristate or oleate as the sole carbon source, providing evidence that S. cerevisiae contains one or more other FAAs, which direct fatty acids to -oxidation pathways (Johnson et al., 1994c).

S. cerevisiae Fas produces acyl-CoAs, while mammalian FAS contains an associated thioesterase known as TEI (Pazirandeh et al., 1989; 1991). The acyl-CoA specificity of TEI determines the nature of the free fatty acids released from FAS. Palmitate is the dominant species generated by TEI-mediated chain termination, while stearate and myristate are minor products. These free fatty acids must be subsequently activated by cellular acyl-CoA synthetases so that they can be targeted to various lipid biosynthetic and degradative pathways. The functional significance of mammalian acyl-CoA synthetases has been assessed using triacsins. All four known triacsins (A-D) (Tanaka et al., 1982; Omura et al., 1986) contain an unbranched chain of 15 non-hydrogen atoms that includes several trans-double bonds and a terminal triazenol group (Fig. 1). Triacsins A and C are competitive inhibitors of the oleoyl-CoA synthetase activity associated with rat liver microsomes (IC = 18 and 9 µM, respectively) (Tomoda et al., 1987). Proliferation of Raji cells in culture is inhibited in a dose-dependent fashion by triacsins (order = C > A > D > B). The degree of inhibition of growth by the various triacsins can be correlated with their ability to inhibit Raji cell membrane-associated long chain acyl-CoA synthetase activity in vitro and by their capacity to inhibit incorporation of oleate into cellular phospholipids and triacylglycerol (Tomoda et al., 1991).

A single long chain human acyl-CoA synthetase cDNA has been recovered to date (Abe et al., 1992). Two long chain acyl-CoA synthetases have been identified in rat. Although limited analysis of their acyl chain specificities revealed no significant differences (Fujino and Yamamoto, 1992), two observations suggest that they may have different physiologic functions. First, they have distinct tissue distributions. Rat liver acyl-CoA synthetase (RLACS) is expressed primarily in liver and adipose tissue (Suzuki et al., 1990), while rat brain acyl-CoA synthetase is expressed primarily in neural tissues (Fujino and Yamamoto, 1992). Second, their responsiveness to perixosome proliferators is different. Fenofibrate increases ACS mRNA levels 6-fold in rat liver (Causeret et al., 1993), while ACS expression in heart and brain appears to be unresponsive to this drug (Schoonjans et al., 1993). Alignments of the primary structures of the four yeast and three mammalian acyl-CoA synthetases indicate that human ACS has greater similarity to rat liver ACS compared with rat brain ACS and Faa2p has greater sequence similarity to the mammalian acyl-CoA synthetases than to any of the other yeast Faas (Johnson et al., 1994c).

The gene products involved in importing fatty acids into S. cerevisiae are unknown. In E. coli, long chain fatty acid transport involves an integral outer membrane protein, FadL, and is coupled to activation by FadD, which is loosely associated with the cytoplasmic face of the bacteria's inner membrane (Black, 1991; Black et al., 1992). A mouse, integral membrane, long chain fatty acid transport protein and an acyl-CoA synthetase have been isolated recently based on their ability to augment uptake of long chain fatty acids into COS-7 cells (Schaffer and Lodish, 1994). The acyl-CoA synthetase has 94% identity with RLACS and a similar tissue-specific pattern of expression. Presumably, it represents the mouse ortholog of RLACS. The finding that fatty acid uptake can be increased by overexpression of either fatty acid transport protein or mouse ACS suggests that both proteins play a role in long chain fatty acid import; fatty acid transport protein may facilitate passage of fatty acids across the plasma membrane, while esterification of imported fatty acids with a polar CoA moiety may prevent their efflux out of the cell (Schaffer and Lodish, 1994).

The large number of FAA genes present in S. cerevisiae raises the possiblity that a given mammalian cell lineage contains multiple acyl-CoA synthetases. This may complicate any assignment of function based solely on the effects of overexpressing a single ACS gene product. The phenotypes produced in S. cerevisiae through deletion of one or more FAA genes provides an opportunity for describing the potential functions of mammalian acyl-CoA synthetases based on their ability to complement faa null alleles. Isogenic FAA1FAA4 and faa1faa4 strains also provide an opportunity to explore the general role of acyl-CoA synthetases in mediating fatty acid import. We have explored these issues in this report.


EXPERIMENTAL PROCEDURES

Strains and Media

The isogenic S. cerevisiae strains YB332 ( MATa NMT1 ura3 his3200 ade2 lys2-801 leu2 FAA1 FAA2 FAA3 FAA4) and YB525 ( MATa NMT1 ura3 his3200 ade2 lys2-801 leu2 faa1::1.9::HIS3 FAA2 FAA3 faa40.3::LYS2) are described in Johnson et al. (1994b and 1994c, respectively).

YPD media consists of 1% yeast extract, 2% peptone, and 2% dextrose. Supplemented minimal media without uracil (SMM-URA) contains 0.67% yeast nitrogen base with ammonium sulfate (Bio 101, Inc.), 2% dextrose, and 0.08% complete supplement minus uracil (Bio 101, Inc.). YPD/agar plates, supplemented with (i) 500 µM fatty acid (NuChek Prep) plus Brij-58 (Sigma; 1% w/v), and/or (ii) 25 µM cerulenin (CER, Sigma), were prepared as described in Johnson et al. (1994b).

To determine the growth characteristics of various strains on YPD, YPD/CER, and YPD/CER/fatty acid plates, equivalent numbers of cells were replica plated using a multiprong applicator (Replaclone, Skatron Instruments). The applicator was dipped into a 1:4 dilution (in 1 M sorbitol) of each culture after the culture had achieved an OD of 1. Duplicate plates were then incubated at 24-37 °C for 3-4 days. All experiments were repeated at least 4 times.

Construction of a Plasmid for Expressing Rat Liver Acyl-CoA Synthetase in S. cerevisiae

pBB358 is a low copy YCp-based plasmid containing the glyceraldehyde-3-phosphate dehydrogenase ( GPD) promoter (Johnson et al., 1994c). pBB360 ( GPD-RLACS) was produced by subcloning a 2.3-kb EcoRI- PstI fragment from pRACS15 (Suzuki et al., 1990) into EcoRI- PstI-digested pBB358.

Metabolic Labeling of Cellular Lipids

Strain YB332 ( FAA1FAA4), transformed with pBB358 (YCp- GPD expression vector without insert), and strain YB525 ( faa1faa4) transformed with plasmids pBB358, pBB330 ( GPD-FAA1, Johnson et al., 1994a;b), pBB365 ( GPD-FAA4, Johnson et al. (1994c)), or pBB360 ( GPD-RLACS), were grown in SMM-URA medium at 24 or 37 °C to an OD 0.8. Cultures were transferred to tubes containing either [9,10-H]myristate, or [9,10-H]palmitate (specific activity = 16 Ci/mmol, 100 µCi/ml culture) and shaken for 1 h at 24 or 37 °C. Cellular lipids were extracted according to Bligh and Dyer (1959), except that 425-600-µm glass beads (Sigma) were added in the initial extraction step. Lipids were resuspended in chloroform/methanol (1:1). Aliquots of 100,000 dpm from each strain were spotted onto silica gel 60 high performance thin layer chromatography (HPTLC) plates (Merck), and separated in a single dimension using methyl acetate, isopropyl alcohol, chloroform, methanol, and 0.25% KCl (25:25:28:10:7). Lipid standards (Sigma) were visualized using iodine vapors. Radiolabeled lipids were detected by spraying the plates with ENHANCE and performing fluorography at -80 °C.

Measurement of Cellular Acyl-CoA Synthetase Activity in Vitro

The myristoyl-CoA and palmitoyl-CoA synthetase activities of cellular lysates from the strains described above were evaluated in an in vitro assay system. Twenty-ml cultures were grown in SMM-URA medium to an OD = 1. Cells were harvested by centrifugation and lysed by vortexing with glass beads in 500 µl of yeast lysis buffer (200 mM Tris, pH 8, 4 mM EDTA, 4 µM pepstatin A, 8 µM leupeptin, 1 mM Pefabloc (Boehringer Mannheim), 4 mM benzamidine, 5 mM -mercaptoethanol, 10% glycerol, and 0.1% Brij 35). The 100-µl acyl-CoA synthetase assays contained 150 mM MOPS, pH 7.1, 2 mM CoA, 2 mM ATP, 10 mM MgCl, 1 mM dithiothreitol, 0.05% Triton X-100, 100 µM EGTA, 50 µM either [9,10-H]myristate or [9,10-H]palmitate (1 Ci/mmol), and cell lysates (concentration, 10-1000 µg of total protein/ml). Following a 15-min incubation at 24 or 37 °C, the reactions were quenched with an equal volume of 5% trichloroacetic acid (prepared in methanol). [H]Fatty acyl-CoA and H-labeled fatty acids were resolved by C4 reverse phase HPLC using an isocratic gradiate of 60% 20 mM potassium phosphate buffer, pH 5.5, and 40% acetonitrile (Knoll et al., 1994). Tritiated products were quantitated using an in-line scintillation counter (model CR; Radiomatic Instruments). Each assay was performed in duplicate and all experiments were repeated at least 3 times.

The Effects of Triacsin C and Cerulenin on Acyl-CoA Synthetase Activities in Vitro

The abilities of triacsin C (Kameda Co.) and cerulenin (Sigma) to inhibit acyl-CoA synthetase activities were evaluated using the in vitro assay system described above. Cellular lysates were prepared from strain YB525 ( faa1faa4) transformed with pBB330 ( GPD-FAA1), pBB365 ( GPD-FAA4), or pBB360 ( GPD-RLACS). Faa1p and Faa2p, each with a carboxyl-terminal tag of 6 histidine residues, were expressed in a fadD strain of E. coli (LS6928 fadRfadD27-zea::Tn 10) (Nunn et al., 1986) and purified to apparent homogeneity using Ni-nitrilotriacetic acid affinity chromatography (Knoll et al., 1994). The 100-µl acyl-CoA synthetase assays contained cell lysate (concentration, 10-1000 µg of protein/ml), or purified Faa1p-6xHis or Faa2p-6xHis (1 µg/ml), [H]myristate (50 µM), plus either 1-100 µM triacsin C (reference control = equivalent concentration of dimethyl sulfoxide (MeSO)) or 100 µM cerulenin (reference control = equivalent amount of ethanol). After a 15-min incubation at 37 °C, the reactions were quenched with an equal volume of 5% trichloroacetic acid/methanol, and products and reactants were resolved by C4 reverse phase high performance liquid chromatography. Each assay was performed in duplicate, and all experiments were repeated 3 times.

The Effect of Triacsin C on the Growth of S. cerevisiae Strains Containing Mammalian Acyl-CoA Synthetases

Strain YB332 ( FAA1FAA4), transformed with the expression vector without insert, and strain YB525 ( faa1faa4), transformed with the vector minus insert, GPD-FAA1, GPD-FAA4, or GPD-RLACS episomes, were grown at 30 °C in SMM-URA to an OD 1. Aliquots (200 µl) were withdrawn from each culture and then added to 4 ml of SMM-URA media containing 0, 1, or 10 µM triacsin C (prepared in MeSO) or MeSO alone, with or without cerulenin (25 µM). Growth rates at 37 °C were monitored every 2 h over the next 12 h by measuring optical density at 550 nm with a Thermo-Max reader (Molecular Devices). Two independent experiments were performed, each in duplicate.

Assay for Fatty Acid Uptake

This assay was adapted from protocols described by Trigatti et al. (1992) for the uptake of oleate in Candida tropicalis and Lai and McGraw (1994) for the uptake of inositol in S. cerevisiae. Uptake was compared in the wild-type strain containing the parental vector and in the isogenic faa1faa4 strain transformed with pBB358, pBB330 ( GPD-FAA1), pBB365 ( GPD-FAA4), and pBB360 ( GPD-RLACS). Cells were grown in SMM-URA media at 30 °C to an OD between 0.5 and 1.0, harvested by centrifugation, and washed once with phosphate-buffered saline, pH 7.4. Cells were resuspended in phosphate-buffered saline to an OD = 10, a 200-µl aliquot of the suspension was withdrawn (10 cells) and added to glass test tubes, and the mixture was prewarmed at 30 °C or chilled to 4 °C for 10 min. Assays were initiated by adding [9,10-H]myristate or [9,10-H]palmitate (dissolved in absolute ethanol; specific activity = 50 mCi/mmol). The final concentration of fatty acid ranged from 5 to 100 µM. The final concentration of ethanol was 0.5% in all reactions. The mixtures were incubated for 0, 30, 60, 120, 300, or 600 s at 4 °C or 30 °C, 10 ml of ice-cold phosphate-buffered saline were added, and cells were collected by passing the suspension through a GF/A glass fiber filter (Whatman). The filters were washed 3 times with 10 ml of ice-cold phosphate-buffered saline, placed in scintillant (Econo-Safe, RPI), and counted. Each time point was assayed in duplicate. Experiments were repeated 3 times.


RESULTS AND DISCUSSION

Rat Liver Acyl-CoA Synthetase Is Functionally Analogous to FAA1 and FAA4

S. cerevisiae strains containing faa1 and faa4 null alleles are not viable at 24-37 °C in YPD media containing 25 µM cerulenin, even when the media is supplemented with 500 µM myristate, palmitate, or oleate (Fig. 2 A plus data not shown). Growth of faa1faa4 cells can be restored by introducing an episome containing FAA1 or FAA4 under the control of the strong GPD promoter (Fig. 2 A). A GPD-rat liver acyl-CoA synthetase episome was also able to sustain growth of faa1faa4 cells on YPD/fatty acid plates when Fas was inhibited by cerulenin (Fig. 2 A). Rescue on YPD/CER/myristate or YPD/CER/palmitate was better at 37 °C than at 24 °C (Fig. 2 A), consistent with the expected temperature optimum of a mammalian enzyme. Complementation by RLACS was also evident when cells were grown in liquid selective media (SMM-URA) containing glucose, myristate, plus cerulenin (Fig. 2 B).


Figure 2: Comparison of the growth rate of a faa1faa4 strain with and without GPD-acyl-CoA synthetase episomes. PanelA, an equal number of cells from YB332 ( FAA1FAA4), transformed with the parental YCp- GPD vector (pBB358), or YB525 ( faa1faa4), transformed with either the parental vector, GPD-FAA1 (pBB330), GPD-FAA4 (pBB365), or GPD-RLACS (pBB360) were plated onto YPD media supplemented with 25 µM CER, with or without 500 µM myristate ( MYR) or palmitate ( PALM). Plates were incubated for 3 days at 24, 30, or 37 °C. Panel B, analysis of the ability of the GPD-acyl-CoA synthetase episomes to rescue growth of faa1faa4 cells in SMM-URA media supplemented with 500 µM myristate and 25 µM cerulenin. Cells were innoculated, during the mid-log phase of their growth at 30 °C, into SMM-URA media or into SMM-URA/MYR/CER and incubated for 12 h at 37 °C. Aliquots were withdrawn at various times after innoculation, and the optical density was determined at 550 nm. The mean ± 1 S.D. is plotted at each time point.



The apparent ability of RLACS to activate imported fatty acids in S. cerevisiae is consistent with the fact that the mammalian cell lineages, which produce this acyl-CoA synthetase (hepatocytes and adipocytes) are normally involved in directing plasma-derived fatty acids to their intracellular lipid metabolic pathways. The ability of Faa1p, Faa4p, and RLACS to activate and direct imported fatty acids to phospholipid biosynthetic pathways was compared directly by analyzing cellular lipids from isogenic FAA1FAA4 or faa1faa4 strains containing GPD-FAA1, GPD-FAA4, or GPD-RLACS episomes. Total cellular lipids were isolated and then fractionated by single dimension HPTLC. The wild-type strain containing Faa1p and Faa4p incorporates label from tritiated myristate or palmitate into phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and phosphatidylcholine when incubated at 24 or 37 °C. This is not the case with the faa1faa4 strain (compare lanes1 and 2 and lanes6 and 7 in Fig. 3, A and B). The failure of the faa1faa4 strain to activate imported fatty acids for phospholipid biosynthesis is associated with a large increase in cellular free fatty acids. Transformation of the faa1faa4 strain with either GPD-FAA1 or GPD-FAA4 restores phospholipid labeling with [H]myristate and [H]palmitate to a level identical to that observed in the wild-type FAA1FAA4 strain ( lanes3 and 4 and lanes8 and 9 of Fig. 3 , A and B). RLACS is also able to direct incorporation of [H]myristate into phospholipids, but the host strain still has a large pool of free fatty acids ( lanes5 and 10 in panelsA and B). Labeling of phospholipids with tritiated palmitate is greater with RLACS at 37 °C (Fig. 3 B) compared with 24 °C (Fig. 3 A). At both temperatures, incorporation of label from [H]palmitate into phospholipid species is lower than that observed with myristate (Fig. 3). These differences in incorporation cannot be ascribed to differences in the efficiency of import of myristate and palmitate since the extent of phospholipid labeling is similar when an isogenic control, containing wild-type FAA1 and FAA4 alleles and the GPD vector without insert, is incubated with either tritiated fatty acid (compare lanes1 and 6 in Fig. 3, A and B).


Figure 3: Metabolic labeling of cellular lipids produced in S. cerevisiae strains containing GPD-acyl-CoA synthetase episomes. Strain YB332 ( FAA1FAA4), transformed with the parental YCp- GPD vector ( lanes1 and 6) and strain YB525 ( faa1faa4), transformed with the the parental vector ( lanes2 and 7), pBB330 ( GPD-FAA1; lanes3 and 8), pBB365 ( GPD-FAA4; lanes4 and 9), or pBB360 ( GPD-RLACS; lanes5 and 10), were grown at 24 °C ( panelA) or 37 °C ( panelB) and labeled with either [H]myristate (C14:0; lanes1- 5) or [H]palmitate (C16:0, lanes6- 10). (the specific activities of the tritiated fatty acids were identical). Cellular lipids (100,000 dpm/lane) were separated by HPTLC in a single dimension, and the plates were subjected to autoradiography for 15 h. The position of migration of lipid standards are shown: NL, neutral lipids; FA, free fatty acids; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine.



The results of these metabolic labeling studies can be correlated with the different growth phenotypes of faa1faa4 ( GPD- episome) cells on YPD/CER/MYR or YPD/CER/PALM plates at 24 and 37 °C (Fig. 2 A) and with the myristoyl-CoA and palmitoyl-CoA synthetase activities measured in lysates prepared from faa1faa4 cells containing GPD-FAA1, GPD-FAA4, or GPD-RLACS (). Lysates prepared from FAA1FAA4 ( GPD-vector) cells during the mid-log phase of their growth had 70-230-fold higher acyl-CoA synthetase activity compared with faa1faa4 lysates when assayed at 24 and 37 °C using myristate or palmitate as substrates ( and data not shown). Since Faa1p and Faa4p together represent 99% of cellular myristoyl-CoA and palmitoyl-CoA synthetase activities in the wild-type strain during exponential growth in glucose-rich media, the low background of endogenous acyl-CoA synthetase activity in faa1faa4 cells provides a convenient assay system for qualitative and quantitative assessment of yeast or mammalian acyl-CoA synthetases produced from GPD-episomes. The myristoyl-CoA and palmitoyl-CoA synthetase activities produced by GPD-FAA4 in faa1faa4 cells are 9-18-fold lower than those obtained with GPD-FAA1. Nonetheless, they are still 40-130-fold greater than in the faa1faa4 strain containing the parental GPD-vector. GPD-RLACS was less effective than GPD-FAA4 in increasing cellular myristoyl-CoA and palmitoyl-CoA synthetase activities; values were 3-21-fold higher than the background levels of faa1faa4 ( GPD-vector) lysates (). While the myrisotyl-CoA and palmitoyl-CoA synthetase activities of faa1faa4 ( GPD-RLACS) lysates are 6-8-fold greater when assayed at 37 °C compared with 24 °C, a much more modest temperature effect (2-fold) was evident with faa1faa4 ( GPD-FAA1) and faa1faa4( GPD-FAA4) lysates ().() In Vitro and in Vivo Sensitivity to Triascin C Provides Evidence for the Functional Similarities of Faa4p and RLACS

In Vitro Studies

Triacsin C is a potent competitive inhibitor of purified Faa2p with a Kof 15 nM using oleic acid (C18:1) as a substrate (Faa2p's Kfor oleate = 25 µM). In contrast, the oleoyl-CoA synthetase activities of purified Faa1p and Faa3p are unaffected by triacsin C in vitro (IC > 500 µM).()

The myristoyl-CoA synthetase activities of faa1faa4 ( GPD-FAA4) and faa1faa4 ( GPD-RLACS) lysates at 37 °C were also strongly inhibited by triacsin C (IC = 4.5 and 6 µM, respectively; ). The myristoyl-CoA synthetase activity in a control faa1faa4 ( GPD-FAA1) lysate was not reduced by 100 µM triacsin C ().

In Vivo Studies

The growth rate of the faa1faa4 strain, with or without the GPD-FAA1, GPD-FAA4, GPD-RLACS, or YCp- GPD episomes, is similar to that of the isogenic FAA1FAA4 strain at 37 °C in SMM-URA media containing dextrose as the fermentable carbon source, 500 µM myristate, but no cerulenin (Fig. 4 A). Thus, when Fas is active, there are no obvious growth differences between the two strains in liquid culture, nor do the episomes produce a deleterious effect. We next defined whether triacsin C could block the rescue by GPD-RLACS, GPD-FAA4, or GPD-FAA1 of faa1faa4 cells at 37 °C in media supplemented with 500 µM myristate and 25 µM cerulenin. Both GPD-FAA1 and GPD-FAA4 produce complete rescue of the faa1faa4 strain throughout the course of a 12-h incubation in SMM-URA/MYR/CER. GPD-RLACS produces partial rescue that is first evident at the 6-h time point. The growth advantage (compared with cells containing YCp- GPD vector alone) becomes progressively more obvious during the next 6 h (Fig. 2 B). This finding provides an indication of the minimum time required to determine if triacsin C affects the activity of RLACS in vivo. Addition of 1 or 10 µM triacsin C had no effect on the growth kinetics of the wild-type FAA1FAA4 strain in SMM-URA/MYR/CER (Fig. 4 B). As expected, faa1faa4 cells containing the YCp- GPD vector minus insert failed to grow under these conditions (Fig. 4 B). Introduction of GPD-FAA1 fully rescued growth in SMM-URA/MYR/CER at 37 °C even in the presence of 10 µM triacsin C (Fig. 4 C). This in vivo result is consistent with the resistance of Faa1p to triacsin C inhibition in vitro (see above). In contrast, triacsin C produces a dose-dependent reduction in growth of faa1faa4 cells containing GPD-FAA4 (Fig. 4 D) or GPD-RLACS (Fig. 4 E).


Figure 4: Effect of triacsin C on the growth of a S. cerevisiae faa1faa4 strain containing various GPD-acyl-CoA synthetase episomes. PanelA, evidence that the episomes have no deleterious effect on the growth of a faa1faa4 strain at 37 °C in SMM-URA media supplemented with 500 µM myristate. Note that cellular Fas activity has not been inhibited by cerulenin in this experiment. Panel B, triacsin C at concentrations up to 10 µM has no effect on growth of a wild-type strain of S. cerevisiae at 37 °C in SMM-URA media containing 500 µM myristate and 25 µM cerulenin. In contrast, the isogenic faa1faa4 strain fails to grow under these conditions. Note that in panelsA and B, growth is monitored as a function of time after innoculation into SMM-URA/CER/MYR. (The innoculum consisted of cells harvested during the mid-log phase of their growth at 30 °C in SMM-URA media minus MYR or CER). Panels C-E, comparison of the ability YCp- GPD episomes encoding Faa1p ( GPD-FAA1; panelC) , Faa4p, ( GPD-FAA4; panelD), or rat liver acyl-CoA synthetase ( GPD-RLACS; panelE) to rescue growth of a faa1faa4 strain at 37 °C in SMM-URA supplemented with 500 µM myristate, 25 µM cerulenin, triacsin C (1 and 10 µM), or an equivalent amount of the dimethyl sulfoxide solvent used to prepare stock solutions of the triacsin (0 µM). Growth is plotted as a function of time after innoculation of exponentially growing cells into the culture media. The mean value ± 1 S.D. is shown.



These results establish that Faa4p and rat liver acyl-CoA synthetase are both inhibited by triacsin C in vivo. A corollary to this conclusion is that triacsin C is able to enter S. cerevisiae during exponential growth and gain access to both of these enzymes. Our data also suggest that RLACS is functionally more analogous to Faa4p than to Faa1p (at least based on their ability to activate imported fatty acids and to be inhibited by triacsin C in vivo). This apparent functional similarity is particularly intriguing given the fact that Faa1p and Faa4p have similar degrees of sequence identity to RLACS (23%, cf. Johnson et al. (1994c)). Analysis of Fatty Acid Uptake in FAA Deletion Strains

Previous studies of Saccharomyces uvarum and Saccharomycopsis lipolytica suggested a saturable mechanism for fatty acid import (Kohlwein and Paltauf, 1983). Uptake of lauric acid (C12:0) into S. lipolytica was not affected by pretreatment with azide even though cellular levels of ATP were reduced to 2% of untreated cells. These results were interpreted to mean that fatty acid uptake occurs by an energy-independent, carrier-mediated process and is not coupled to fatty acid activation (Kohlwein and Paltauf, 1983).

The availability of S. cerevisiae strains with wild-type and null FAA1 and FAA4 alleles allowed us to define the effects of Faa1p, Faa4p, and RLACS on the uptake of C14:0 and C16:0. PanelsA and B of Fig. 5 show the time-dependent changes in cell-associated radioactivity obtained when the FAA1,FAA4 strain containing the GPD vector was incubated at 30 °C with 10 or 100 µM fatty acid. Uptake was linear for the first 60 s at both concentrations for both fatty acids. Studies conducted at 4 and 30 °C with 100 µM fatty acid indicated that uptake of C14:0 and C16:0 was temperature-dependent (Fig. 5 C and data not shown). The concentration of C14:0 or C16:0 in the assay mixture was varied from 5 to 100 µM, and uptake was monitored at 30 °C after a 30-s incubation ( i.e. the midpoint of the linear phase). The results (Fig. 5 D) indicate the S. cerevisiae contains a saturable fatty acid transport system with K = 55 and 46 µM for C14:0 and C16:0, respectively. These calculated Kvalues are similar to those reported for oleate in C. tropicalis (56 µM; Trigatti et al., 1992).


Figure 5: Fatty acid uptake in isogenic FAA1FAA4 and faa1faa4 strains. PanelsA and B, time course of uptake of myristate (A) or palmitate ( B) into FAA1FAA4 cells containing the parental YCp- GPD vector. The y axis on the left side measures cell-associated radioactivity when the concentration of fatty acid in the assay was 100 µM. The y axis on the right side of these two panels shows the values obtained when [fatty acid] = 10 µM. Panel C, uptake of myristate into FAA1FAA4 cells containing YCp- GPD at 4 and 30 °C. Panel D, fatty acid uptake into FAA1FAA4-YCp- GPD cells after a 30-s incubation at 30 °C in the presence of 5, 10, 20, 50, or 100 µM C14:0 or C16:0. Panels E-G, uptake of C14:0 or C16:0 into (i) the FAA1FAA4 strain transformed with the parental YCp- GPD vector and (ii) the isogenic faa1faa4 strain transformed with the parental vector, GPD-FAA1, GPD-FAA4, or GPD-RLACS. The fatty acid and its final concentration are described in each panel. The mean ± 1 S.E. is plotted at each time point for each strain. Note that introduction of GPD-FAA1, but not GPD-FAA4 or GPD-RLACS, reduces import of C16:0 2-fold. No effect is noted on C14:0 import. The significance of the GPD-FAA1 effect remains unclear although it is reproducible ( n = 3 experiments, each done in duplicate).



Isogenic FAA1FAA4 and faa1faa4 strains containing YCp- GPD vector, GPD-FAA1, GPD-FAA4, or GPD-RLACS, were incubated with C14:0 or C16:0 at 30 °C. Uptake was assayed at two concentrations of fatty acid: 10 µM (subsaturating) and 100 µM (saturating; defined according to Fig. 5D). We used two concentrations because we were concerned that at high levels of fatty acid, the rate of transport may exceed the catalytic capacity of the acyl-CoA synthetases and therefore the effects of the latter on transport could be negligible. Conversely, at lower concentrations, metabolism of the fatty acids may function as kinetic trap that displaces the transporter from equilibrium and thereby effectively couples the rate of transport to acyl-CoA formation. The isogenic FAA1FAA4 (YCp- GPD) and faa1faa4 (YCp- GPD) strains exhibit no significant differences in their ability to transport myristate and palmitate when fatty acid is present at either 10 or 100 µM (Fig. 5, D- G and data not shown). Moreover, addition of GPD-FAA1, GPD-FAA4, or GPD-RLACS to the faa1faa4 strain produces no increase in the import of either fatty acid (Fig. 5, D- G).

These findings, together with two other observations described above ( i.e. (i) Faa1p and Faa4p together account for 99% of the myristoyl-CoA and palmitoyl-CoA synthetase activities in this yeast and (ii) faa1faa4 (YCp- GPD) cells accumulate free C14:0 and C16:0 ( cf. lanes2 and 8, in Fig. 3 , A and B)) suggest that import and activation of myristate and palmitate are not coupled processes in S. cerevisiae. Prospectus

The results of these genetic complementation studies suggest a number of experimental systems that may be useful in the future. First, the dependence of the faa1faa4 strain on rat liver acyl-CoA synthetase for growth in YPD/CER/MYR or SMM-URA/CER/MYR media represents a biological assay for identifying inhibitors of this mammalian enzyme. Such inhibitors may have therapeutic uses. Second, the faa1faa4 strain and these growth conditions could also be used to isolate cDNAs encoding acyl-CoA synthetases that are responsible for activating imported fatty acids in other species ( e.g. human, various human pathogens). Selective or nonselective inhibitors of these enzymes could then be identified with the biological assay. Finally, the functions of other types of mammalian acyl-CoA synthetases as well as their interactions with fatty acid transport proteins may be more readily definable, in exponentially growing or stationary phase (Werner-Washburne et al., 1993) S. cerevisiae than in their cells of origin. This belief is based on ease of genetic manipulation of S. cerevisiae plus the availability of isogenic strains with various combinations of faa alleles and well-characterized mutations that affect enzymes and/or organelles that participate in lipid metabolism ( e.g. peroxisomal assembly ( PAS) mutants; Erdmann et al., 1989; 1991).

  
Table: 0p4in Not determined.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health (AI27179 and AI30188) and Monsanto. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Molecular Biology and Pharmacology, Box 8103, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-7243; Fax: 314-362-7058; E-mail: jgordon@pharmdec.wustl.edu.

The abbreviations used are: Fas, yeast fatty acid synthetase; Faap, S. cerevisiae fatty acid activation protein; FAA, S. cerevisiae fatty acid activation gene; FAS, mammalian fatty acid synthetase (acyl-CoA:malonyl-CoA C-acyltransferase (decarboxylating, oxoacyl- and enoyl-reducing and thioester-hydrolyzing); EC 2.3.1.85); RLACS, rat liver acyl-CoA synthetase; CER, cerulenin; HPTLC, high performance thin layer chromatography; ACS, acyl-CoA synthetase; MOPS, 3-( N-morpholino)propanesulfonic acid.

Ole1p, a cis desaturase, accounts for all de novo production of monoenoic fatty acids from saturated acyl-CoA substrates in S. cerevisiae (Bossie and Martin, 1989; Stukey et al., 1989, 1990; McDonough et al., 1992).

Addition of 100 µM cerulenin to lysates prepared from faa1faa4 strains containing GPD-FAA4, GPD-FAA1 or GPD-RLACS had no significant effect on their myristoyl-CoA and palmitoyl-CoA synthetase activities, providing further evidence that cerulenin's inhibitory effect on acyl-CoA synthesis occurs via its effects on Fas rather than on Faa1p, Faa4p, or RLACS.

L. J. Knoll, G. W. Gokel, and J. I. Gordon, in preparation. (Note that C18:1 was chosen because it is a substrate for purified Faa1p, Faa2p and Faa3p (Knoll et al., 1994a).)


ACKNOWLEDGEMENTS

We thank Tokuo Yamamoto (Tohoku University Gene Research Center) for generously supplying the cDNA for rat liver acyl-CoA synthetase and George Gokel for insights about the structural similarities between triacsins and cerulenin.


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