A defect in the sterol : Steryl ester interconversion in a mutant of the yeast, Saccharomyces cerevisiae

Biochimica et Biophysics Acta, 1123 (1992) 127-132 0 1992 Elsevier Science Publishers B.V. All rights reserved

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127 0005-2760/92/$05.00

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A defect in the sterol : steryl ester interconversion in a mutant of the yeast, Saccharomyces cerevisiae George A. Keesler, Scott M. Laster and Leo W. Parks Department of Microbiology; North Carolina State Unir~ersity;Raleigh, NC (U.S.A.) (Received

Key words:

12 July 1991)

Sterol; Trafficking;

Yeast; Mutant

A culture cycle dependent interconversion of sterols and steryl esters is disturbed in a mutant of Succharomyces cerevisiue. Independent extragenic suppressors to this mutant return the mutant’s pleiotropic phenotype to that of

the parental wild type. Concomitant with the alterations in interconversion, modifications were found in the yeast proteins that antigenically react with antibodies elicited against mammalian apolipoproteins. Suppressor mutations returned the aberrant immunoblot banding pattern of the mutant to that of the wild type in apolipoprotein B.

Introduction Steryl esters in yeast are found principally distributed as an approximate 50:50 mixture with triacylglycerol in protein-enclosed microdroplets [l]. While these esters are not used for carbon and energy during starvation of the cells, they are metabolically interconvertible with free sterols, depending on the stage of the culture cycle of the organism 121.Sterols are hydrolyzed from the steryl esters early in the culture cycle to accommodate the organism’s need for free sterol for nascent membrane formation. Extensive re-esterification occurs when the culture enters stationary phase following active growth. The orderly discrimination, transport, and distribution of cellular components in response to physiological demands for those substances has been termed ‘trafficking’. Models for understanding the regulation of the partitioning and reversible vectorial concentration of the lipids in trafficking are lacking. Mutants defective in the process could be helpful in developing an understanding of the mechanisms involved. By dissecting aberrant partitioning patterns effected by muta-

tion it may be possible to develop experimentally amenable models. We report here an organism, previously isolated for its ability to accumulate exogenously supplied sterols [3], which has a defect in the sterohsteryl ester interconversion. Extragenic suppressors of that mutant have been isolated which return the phenotype to that of the parental wild-type (Keesler, Lorenz, and Parks, in preparation). We have reported previously that yeast have proteins that antigenically cross-react with antibodies elicited against mammalian apolipoproteins [4]. However, we were unable to assign any physiological role for those proteins. We have now observed that the defective interconversion of sterols and steryl esters and subsequent modifications in the suppressed clones are accompanied by alterations in those proteins. Materials and Methods Strains

Yeast strains of Saccharomyces this study are listed in Table I.

cerevisiae used in

Growth conditions Abbreviations: PPO, (2,5-diphenyloxazole); POPOP, (7,4-bis-(5phenyloxazole) benzene); PMSF, (phenylmethylsulfonyl fluoride); BSTFA. fbis (trimethylsilyl)-trifluoroacetamide); FLL, floating lipid layer; PAGE, polyacrylamide gel electrophoresis. Correspondence: L.W. Parks, Department of Microbiology; Carolina State University; Raleigh, NC 27695-7615, U.S.A.

North

Cultures were grown on rich medium (YPD; 2% peptone, 1% yeast extract, 2% dextrose) or in defined medium (YNB), containing 0.6% yeast nitrogen base without amino acids, 2% dextrose, 0.6% succinate, 50 pgg/ml each of tyrosine, phenylalanine, leucine, lysine, tryptophan, histidine, uracil and adenine; methionine

12X

Strain

Genotype

Source

?
MATa MATa MATa MATtv MATa MATa MATn

YGSC ~’ Lewis [3] Thi\ study This study This study This study This study

SUC’ r~cllgul2 CUPI qx’-I SUEI-I SUEZ-I 1rpc7-I are/- I .xtr’_7-I r(p(.Z-I SUEI-I SUEZ-I upc’- I .s~u’ I I SUE’- I ~qx?- I SUE I-I WC’-I 1qx.1- 1 SIICI - I r~2- I

of rcferrnce

” ” ‘I ”

” YGSC. Yeast Genetic Stock Center, Berkeley, California. ” Ohtained from a cross hctwcen parentals Llpc3) and Sue RZ.

was added to a final concentration of 100 pug/ml. Cultures were grown in 250 ml shaker flasks at 30°C with aeration. Cell density was determined, using a Klett calorimeter. to be proportional to Klett units according to the formula: log cells/ml = (log Klett + 1.86)/0.6486, as confirmed by direct particle enumeration using an Elzone electronic cell counter. Stcrol analysis Cell samples were collected by centrifugation and washed with 0.5% potassium acetate. Cell pellets were frozen, lyophilized and the sterols were extracted as described previously [5]. A known amount of cholesterol was added as an internal standard in order to determine extraction and recovery efficiencies of each sample. Sterols were derivatized by the method of Thompson et al. [61. Separation and quantitation of stcrols was by capillary gas chromatography in a Hewlett Packard 5850 instrument equipped with a fused Silica SPBl capillary column (Supelco. Bellefonte, PA) of 0.25 pm film thickness, and 30 m X 0.32 mm i.d. column dimensions. Chromatography was performed with column oven at 230°C and injector and detector ovens at 280°C. Carrier gas was helium at 32 psi and make up gas was nitrogen. Data were recovered and processed with a Waters Maxima 820 data acquisition and processing program (Milford, MA) on an IBM/AT work station. Amount of sterol was expressed in pg sterol/mg cell dry weight. Intercon~~ersionof sterols Representative strains were grown to stationary phase in [“CH,]methionine (0.6 kCi/ml, 100 pg/ml) minimal medium according to the method of Taylor and Parks [2]. C-28 sterols (principally ergosterol) are the only components in the non-saponifiable fraction of yeast labelled with the methyl group of methionine. The cultures were then washed with fresh medium and diluted 1 : 100 into fresh unlabelled medium. Growth was monitored, and samples were taken at the indicated time intervals. Cells were collected and washed by centrifugation. frozen and lyophilized; the sterols

were extracted following the Me,SO or alkaline saponification procedures of Parks et al. [7]. Separation of fret stcrols and steryl esters was achieved by thin layer chromatography using the method of Skipski et al. [Xl. Authentic standards wcrc run of free and cstcrificd sterols. Representative bands were scraped and resuspended in toluene-PPO (2,S-diphenyloxazole)-, POPOP (7,4-his-(S-phenyloxazolc) benzene) scintillation cocktail [3]. Radioactivity was measured in ;I Beckman model LS 5X01 scintillation counter (Fullerton, CA). Each analysis was performed in triplicate. Interconversion of sterols was detcrmincd on a DPM/ml basis. Protein isolation from the floatirly lipid luger (FLL) The cellular floating lipid layer was isolated as previously described 141. Protein content was determined by the Pierce BCA assay (Pierce Chemical Co., Rockford, IL). Polyacrylarnide gel electrophoresis (PAGE) and Westem blot analysis SDS PAGE was performed on samples at room temperature [9l. Approximately 20 pg/ml of protein from both crude and FLL extracts were loaded into each well. Bands were visualized using Coomassic brilliant blue R-250 or silver staining by Bio Rad silver stain kit. Proteins were transferred to nitrocellulosc membranes at 80-100 v and approximately 260 mA at 4°C for 4 h. Western blotting was performed by using rabbit anti-rat apolipoproteins (Al, B and E) as the primary antibodies; these were a gift from Dr. David Usher, University of Delaware. Secondary antibody was goat anti-rabbit linked to horseradish peroxidase and effective antigen:antibody reactions were visualizcd using the TMB membrane peroxidase substrate system. both from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD). A control of normal rabbit serum was used to detect any nonspecific binding. C’iwtnicals All chemicals were reagent grade and the solvents were redistilled before use. [ “CH,]Methionine was from New England Nuclear Corp. (Wilmington, DE). Constituents for the growth media were from Difco (Detroit, MI). Proteins for molecular weight standards were from Bio Rad (Richmond, CA). Ergosterol, cholesterol, tyloxapol. Tween-30, PPO, POPOP, PMSF. and apolipoproteins were from Sigma Chemical Corp. (St. Louis, MO). BSTFA and trimethlychlorosilanc were purchased from Supelco (Bellefonte, PA). Results and Discussion Yeast strain Upc20 was shown to accumulate a large amount of steryl ester, in comparison to the wild type

129 TABLE

II

Endogenous

ergosterol

content

and distribution

into the free sterol and

steryl ester fractions

Quantitation of sterols was by gas chromatography on a 30 m SPBI capillary column (Supelco) after TLC, and as described in Materials and Methods. Steryl esters were converted to free sterols via alkaline saponification. Cholesteryl palmitate was used as the internal standard for extraction efficiency. Values represent + S.D.‘s for triplicate determination of each sample for each experiment. (n), number of experiments. Strain

X2180-1A UPC20 Sue R2 SUE 5A SUE 5B SUE 5C SUE 5D

(n) 4 4 3 2 2 2 2

Free sterol

Steryl ester

yS/mg

kCLg/mg dry wt.

dry wt.

2.07 + 0.35 2.66 k 0.34 2.19kO.56 1.95kO.22 2.06kO.4 2.35 k 0.25 2. I3 * 0.33

3.13+0.40 12.1 +2.75 3.12f0.94 10.5 f 1.21 6.54 f 0.87 5.94kO.45 3.30 f 0.50

[3]. We have isolated Sue R2, a suppressor variant of Upc20, that returned the pleiotropic characteristics of the mutant to the wild type phenotype, and a genetic analysis has been performed (Keesler, Lorenz, and Parks, in preparation). When the suppressor strain was back-crossed to Upc20, the resulting segregation pattern indicated that two suppressor genes were involved. We undertook an analysis of those segregants for free and esterified sterol, and compared those data with the appropriate parental strains. The results, shown in Table II, are data from representative isolates from the genetic analysis. The distribution of sterol into the free fraction remains relatively constant between all strains tested. Thus, the ability to maintain a bulking free sterol level is not altered. However, the greatest disparity between all strains occurs in the steryl ester fractions. As reported previously [3], UpQO consistently accumulated a high amount of steryl esters. Progeny of a cross between parentals UpQO and Sue R2 showed wild type levels of steryl esters in strain Sue 5D, when both suppressors segregated in an otherwise upc2 background. When neither suppressor was present (strain Sue 5A), UpQO levels of steryl esters returned as was expected. Interestingly, when the suppressors segregated independently from one another, only partial or intermediate levels of steryl esters accumulated (strains Sue 5B and 50. In a previous report from our laboratory we demonstrated that wild type yeast, entering the stationary phase, package excess ergosterol into the ester fraction [lo]. As the cell culture is transferred to fresh medium and growth is initiated, the esters are rapidly hydrolyzed and repartitioned into the free sterol fraction [2]. In effect, hydrolysis of the ester fraction supplements the endogenous free ergosterol synthesis early in expo-

nential phase of the cell growth. As the culture again reaches the end of active growth, large amounts of ergosterol are transferred to the ester pool. Thus, a normal esterification:hydrolysis cycle appears to be important in maintaining an adequate free ergosterol flux within the cell. We asked if normal partitioning of sterol into and out of the ester fraction occurred in Upc20 and what role the suppressor mutations had in the interconversion of sterol. Cells were pregrown to stationary phase, and the sterols were labelled at C-28 with [‘4CH,]methionine. The cultures after reaching stationary phase were reinoculated into fresh growth media without additional radiolabel and samples were harvested at various time intervals during growth. Fig. 1 shows this interconversion of sterols during the early part of the culture cycle for the parental X2180-la (panel A), UpQO (panel B), Sue 5B (panel 0, and Sue 5D (panel D>. With the initiation of cell growth there is a rapid hydrolysis of steryl ester in the wild type, and the labeled sterol is found in the free fraction. A very different pattern is observed with the mutant Upc20. Virtually no interconversion of sterol occurs, although a similar culture cycle is observed. Furthermore, one suppressor alone (Sue 5B) is not sufficient in re-establishing normal trafficking. Interestingly, when both of the suppressor alleles are present (Sue 5D), normal interconversion of sterol is returned. Our experiments reported here demonstrate the interconversion is modified in the mutant. Although we have shown the presence of steryl ester synthetase and steryl ester hydrolase activities in UpQO [3], the defect in Upc20 appears to be an inability to mediate normal sterol trafficking into and out of the ester fraction, Therefore, some component appears to prevent the free/ester interconversion which occurs in the wild type parental and is re-established in a double suppressor background. In higher eucaryotes apolipoproteins are involved in the mobilization, transport, and esterification of sterol [l l-131. Yeast proteins reactive to antibodies elicited against apolipoprotein Al, B and E have been reported [41. It occurred to us that the defect in trafficking might be due to modifications in one or more of these proteins that display cross-reactivity to mammalian apolipoproteins. A crude fraction and a lipid rich fraction ‘floating lipid layer’ (FLL) were obtained from the wild type, Upc20 and Sue R2. Proteins from them were separated by SDS PAGE, and blot transferred to nitrocellulose membranes. They were reacted with purified rabbit anti-rat apolipoprotein IgG and with goat anti-rabbit antibody linked to horseradish peroxidase. The wild type, Upc20, and the strain carrying both suppressor genes each expressed proteins which reacted with antibodies against rat apolipoprotein B, although the pat-

130 tern of reactivity was different in each strain (Fig. 2). In crude fractions isolated from the wild type the antibody reacted primarily with three proteins between 70-85 kDa (76, 78, and 84.5 kDa). The antibody also showed weak cross-reactivity with proteins at approximately 42 kDa, 57 kDa and a high mw. faction ( > 100 kDa) which failed to enter the gel effectively. In the mutant, Upc20, most of the reactive material in the 70-85 kDa range was lost, while increases in reactivity were noted in the high m.w. fraction and the 42 kDa band. The suppressor, Sue R2. gave a reaction pattern similar to both X2180-la and Upc20. The majority of the cross-reactive material was in the high molecular

weight fraction as was seen in Upc30. but unlike Upc20, there was no increase in the reactivity at 42 kDa. Most of the yeast steryl esters are contained in protein coated low density microdroplets, which form the floating lipid layer during centrifugation. Protein preparations from the FLL differed in some aspects from the crude preparations in reactivity with the antiapolipoprotein B antibody. The reactions with higher molecular weight material observed in crude preparations disappeared. Furthermore. the increased reactivity at 32 kDa in CJpc30 was lost in the FLL fraction. However, Upc20 showed almost complete loss of the 76 kDu band which was present in the wild type and

7w D Eao-

500 1 400 I 300 c 200 0 100, 10

Time

0

20

200

100

i 0

10

20

Time

(hours)

Time

(hours)

IO

Time

(hours)

30

E

O-l

0

10

Time

20

30

20

30

(hours)

(hours)

20 Fig. 1. The interconversion of esterified and free sterol as measured during aerobic growth. Cell cultures of X7180 and Upc20. SW 5B, and Sue 5D were grown to stationary phase in minimal medium with the addition of [‘“Clmethionine. The cells were washed and inoculated (1: 100 dilution) into fresh medium without labeled methionine. Growth (A) was monitored (A) X2180. (a) Upc20. (0) Sue 5B, (0) Sue SD, and samples were taken at specific time intervals throughout the cycle until growth was completed: (B) (0) X2180, free sterol. Cm) X2180, stetyl ester. (C) ( 0 ) Upc20 free sterol: Upc20 steryl ester; (D) ( q ) Sue 58, free sterol, ( n ) Sue 5B steryl ester; (E) ( q ) Sue 5D. free sterol. and ( w ) Sue 5D stetyl ester.

131 A

kDa kDa 97 * 66 *

31*

123123 Fig. 2. Western blot analysis demonstrating the cross reactivity between yeast protein and rat apolipoprotein B antibodies. Approximately 20 pg of protein from either crude or FLL extracts were loaded into each well. Lanes: (A) CRUDE (1) X2180 crude fraction (2) UpQO crude fraction, (3) Sue R2 crude fraction; (B) FLL (1) X2180 FLL fraction, (2) Upc20 FLL fraction, (3) Sue R2 FLL fraction.

partially re-established in the complemented suppressor. Western blot analysis with rat apolipoprotein E antibodies showed reactivity with only one protein of an approximate size of 71.5 kDa in either crude or FLL preparations (Fig. 3). However, in the crude fractions the degree of cross-reactivity was much greater in both Upc20 and Sue R2 when compared to the wild type. This pattern was not observed in FLL preparations. Anti-apolipoprotein Al antibodies reacted with a single 35 kDa protein in both crude and FLL prepara-

B

A

kDa 97 -b 66 * 454 ,,

31-

.A&

Fig. 4. Western blot analysis demonstrating the cross reactivity between yeast protein and rat apolipoprotein Al antibodies. Approximately 20 pg of protein from either crude or FLL extracts were loaded into each well. Lanes: (A) CRUDE (1) X2180 crude fraction (2) Upc20 crude fraction, (3) Sue R2 crude fraction; (B) FLL (1) X2180 FLL fraction, (2) Upc20 FLL fraction. (3) Sue R2 FLL fraction.

tions (Fig. 4). No differences in the patterns of reactivity were noted among the different strains tested. Human apolipoprotein B is a glycoprotein that is found in two forms, B-100 (549 kDa) and B-48 (264 kDa) [14]. Apolipoprotein B is the major protein component of the low density lipoproteins (LDL) of blood plasma and, like apolipoprotein E, a constituent of the very low density lipoproteins (VLDL) [15]. LDL is the principle acceptor of steryl ester in plasma [16]. Patients with non-insulin dependent diabetes mellitus have a defect in cholesteryl ester transfer that is associated with the diabetic VLDL and LDL [17]. It is interesting that the upc20 mutant also appears to have a defect in steryl ester mobilization. The exact relationship between the defect in interconversion and the altered proteins can not be established from these experiments. They do provide a first step in attempting to define sterol trafficking in the simple eucaryote. The apolipoprotein-like proteins may serve as a carrier of the ester or be a component of the esterification/ hydrolysis enzymes. These are the subject of continuing studies in our laboratory. Acknowledgements

I. .:

*

)_

‘j.

123123 Fig. 3. Western blot analysis demonstrating the cross reactivity between yeast protein and rat apolipoprotein E antibodies. Approximately 20 pg of protein from either crude or FLL extracts were loaded into each well. Lanes: (A) CRUDE (1) X2180 crude fraction (2) Upc20 crude fraction, (3) Sue R2 crude fraction; (B) FLL (1) X2180 FLL fraction, (2) Upc20 FLL fraction, (3) Sue R2 FLL fraction.

This research was supported in part by grant DCB8814387 from the National Science Foundation, and U.S. Public Health Service Grant DK37222 from the National Institutes of Health, and the North Carolina Agricultural Research Service (Raleigh, NC 276957643). The authors gratefully acknowledge the helpful discussion and assistance in manuscript preparation that was provided by Dr. Kelly Tatchell.

References Clausen, M.K., Christansen, K.. Jensen. P.K. and Bchnke. 0. (1974) FEBS Letters 43. 176-17Y. Taylor. F.R. and Parks. L.W. (197X) J. Bacterial. 136, 531-537. Lewis. T.A.. Keesler, G.A.. Fenner. G.P. and Parks. L.W. (1988) Yeast 4, 93-106. Keesler, G.A.. Moore. S., Usher. D.C. and Parks. L.W. (IYYI) Biochem. Biophys. Res. Commun. 174, 631-637. Casey. W.M., Burgess, J.P. and Parks. L.W. (IYYI) Biochim. Biophys. Acta 1081, 279-284. Thompson Jr.. R.H., Patterson, G.W. and Thompson. M.J. (IYXI ) Lipids 16, 694-69’). Parks, L.W., Bottema. C.D.K.. Rodriguez. R.J. and Lewis, T.A. (1985) Methods Enzymol. 111, 333-346. Skipski. G.P.. Smolowe. A.F.. Sullivan, R.C. and Braclay. M. ( IYhS) Biochim. Biophys. Acta 106, 386-31~6.

Y Lammrlli. U.K. (1970) Nature 717. hXtI-6X5. IO Bailey. R.B. and Parks, L.W. (1974) J. Bacterial. 114. hOh-012. I I Barharas. R.. Puchois. P.. Grimaldi. P.. Barkia, A.. Fruchart, J. and Ailhaud. G. (19x7) Biochem. Biophys. Res. Commun. I-lY. 545-553. 12 Ogawn, Y. and Fielding, C.J. (IYHS). Method Enzymtrl. I I I, 774-7x5. I3 Busch. S.J.. Duvic, C.R.. Ellsworlh. J.L.. Ihm. J. and Flarmony. J.A.K. (IYXh) Anal. Biochem. 153, 17%1% II Kane. J.P.. Hardman. D.A. and Paulus. H.E. (IYXO) Proc. Natl. Acad. Sci. USA 77. 746%246’). I5 Breslow. J.L. (IYXS) Annu. Rev. Biochem. 54. 6YY-777. I6 Fielding. C.J. and Fielding. P.E. (IYXI) J. Biol. (‘hem. 256. 2102-‘7104. 17 Fielding. C.J.. Rraven. G.M.. Liu, Ci. and Fielding. P.E. (IYXJ! Proc. Natl. Acad. Sci. LISA X1. 2512-25lh.