Isolation of Chinese hamster cell mutants defective in the receptor-mediated endocytosis of low density lipoprotein

J. Mol.

12iol. (1981) 150. 167-184

Isolation of Chinese Hamster Cell Mutants Defective in the Receptor-mediated Endocytosis of Low Density Lipoprotein

l)Qpnrtwwnt I’nitwsity

of Mol~otlnr (hetics and ~ntrrnal Mrdicine. of TexaR Health Scieme Cerlter nt Dnllrrs. Dallas. Texas 752.35. r’.~y..d

This paper describes a procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of the unique structure of low density lipoprotein, a plasma cholesterol transport protein that enters cells by receptor-mediated endocytosis. l,Dl,$ contains a core of cholesteryl cstcr that can be extracted and reconstituted with hydrophobic molecules that ceonvert the LDL into a toxic or fluorescent particle. Mutagenized Chinese hamster ovary cells were incubated with reconstituted LDL containing toxic 25. hpdroxycholcstcryl oleate. Wild-type cells take up this lipoprotein aia the LDL recept,or, liberate the 2Shydroxycholesterol in lysosomes. and die. To identif? c,olonies of receptor-deficient cells from among the few survivors of the first selection step, we incubated the cells with LDL reconstituted with a fluorescent cholestervl ester and picked colonies that failed to accumulat)e fluorescence. The two-step*isolatJion procedure yielded receptor-deficient cells at a frequency of 1 in I05. The mutant cells grew in the presence of LDL reconstituted with 2.ihydroxycholesteryl oleate at concentrations 100.fold higher than those that kitled parental cells. The altered phenotypes have remained stable for more than 200 population doublings under non-selective conditions. Inasmuch as LDL can be c*oupled to ligands that bind to receptors other than the LDL receptor, the above method may have general utility in isolating cells with mutations affecting other rrc.eptor systems.

1. Introduction Receptor-mediated endocytosis is a process by which physiologically active macromolecules, such as transport

animal cells take up proteins and peptidr

t Present address: Whitaker College and Kiology Department, Mrtssachuwtts In&ituk <,f ‘I’ec~hnology. (‘ambririge. MA 02139. L1.S.A. : Abbreviations used : LDL. low density lipoprotein: CHO cdls. Chinese hamster ovary cells: PMC’A oleate: EMS, ethylmethane sulfonate: oleate. :~-p~renemethvl-~3.24-dinor-5-rholen-~-oate-~~-~l IJ’DS. lipoprotein-tle~~ient serum; HMG Co.4 reductase, 3-h~tlros~~3-meth~l~lntar~l CoA retiwtxse. 167 (922-2836/81/220167~ 1X $02.00/0 P 1981 i\rademir Press Inc.. (Lonclcm) I,td.

168

M. KRIEGER.

M. S. BKO\VN

ANI)

.I. I,. GOLDSTEIS

hormones, from extracellular fluid. The macromolecules bind to receptors on the surface of cells, after which the portions of the plasma membrane bearing the receptor-ligand complex, called coated pits, inraginate and pinch off from the cell surface to form endocytic vesicles. The ligand-containing vesicles are then directed t’o sites in the cell, frequently lysosomes. for further processing (for a review. see Goldstein it al.. 1979n). One of the best understood systems of receptor-mediated endocytosis is the low density lipoprotein receptor pathway (Brown Xr. Goldstein, 1979). This pathway consi& of a co-ordinated sequence by which cells bind LDL$, the major transport protein for cholesterol in human plasma, and internalize it by receptor-mediated endocytosis. The hydrolysis of the LDL-bound cholester.yl esters in lysosomes synthesis and growth. supplies cells with the cholesterol required for membrane Mutations in the LDL receptor pathway have been identified in diploid fibroblasts cultured from patients with a genetic disease called familial hypercholesterolemia (Goldstein & Brown, 1979). These cells have defects in the binding or in delineat,ing the separate roles internalization of LDL ; they have been invaluable of binding and internalization in mediating the delivery of cholesterol to cells. The further usefulness of these human mutations in the understanding of receptor-mediated endocytosis is limited for several reasons. First, diploid fibroblasts, which have a finite life-span in culture. cannot readily be cloned and therefore do not) lend themselves to manipulation by somatic cell genetic techniques. Second, the variety of mutations available in human material is limited. Third, naturally occurring mutations in receptor-mediated endocytosis of ligands other than LDL have not been described. For these reasons. it seems desirable to develop techniques that, permit’ the isolation of clones of established cell lines that bear mutations in receptor-mediat,ed endocytosis. In the current studies, we report a two-step procedure for isolation of mutant CHO cells with defects in the LDL receptor pathway. The selection protocol takes advant,age of a unique property of LDL, namely its hydrophobic core. This core, which is normally composed of about, 1500 molecules of cholesteryl ester. can be removed with heptane and the lipid-depleted lipoprotein can be reconstituted with a variety of exogenous lipids, including toxic and fluorescent, compounds (Krieger rt al., 197&J, 1979). These lipids will not enter cells in large amounts unless the LDL is taken up by receptor-mediated endocytosis. In the first selection step. mutagenized cells are incubated with LDL that has been reconstituted with a toxic compound (25hydroxycholesteryl oleate) so that cells expressing a normal LDL receptor pathway will internalize the particle. deliver it’ to lysosomes, liberate the toxic compound, and die. In the second step. the ~11s surviving the first selection are incubated in the presence of a fluorescent reconstituted LDL and those cells that fail to take up and accumulate the fluorescent dye, as determined microscopically, are isolated and characterized. This second step eliminates cells that survive the first’ selection because they are resistant to the toxic compound. procedure, we have isolated mutant clones of (‘HO cells that Using this two-step show markedly deficient LDL receptor activity.

LDL

RECEPTOR

MVTATIOSS

IS

(‘HO

(“ELLS

I69

2. Materials and Methods (a) Materials Sodium nL-mevalonate was prepared from nL-mevalonic acid lactone (Sigma Chemical (‘0.) as previously described (Brown et al., 1978). Compactin (ML-236B) was a gift from DI Akira Endo and was used as the sodium salt (Brown et al., 1978). [ li4C]oleic acid (56 mCi/mmol) and sodium [ ‘251]iodide (16 mCi/pg) were purchased from Amersham/Srarlr. nr,-3-Hydroxy-3-methyl-[3-‘4(l]glutaryl Coenzyme A (56 mCi/mmol) and 13H]sucrosc~ (I 1.2 Ci/mmol) were obtained from New England Nuclear Corp. 25-Hydroxycholesteryl oleate and 3-pyrenemethyl-23,24-dinor-5-cholen-22-oate-3~-yl oleate were synthesized by DI .I. R. Falck in this laboratory. Ethyl methanesulfonate was purchased from Sigma Chemical (‘0. Fetal calf serum and newborn calf serum were obtained from Flow Laboratories and (irand Island Biological Co., respectively. Phosphate-buffered saline (cat. no. 310-4190). Ham’s F-12 medium (cat. no. 430-1700) and 905% trypsin/002°/b EDTA solution (cat, no. 619.X)50) were purchased from Grand Island Biological Co. All other materials were olnained from sourres as previously described (Goldstein et al.. 1976: Kriegrr PI al., 1978e.h) (b) Lipoproteins Human LDL (d 1.019 to I.063 g/ml) and human lipoprotein-deficient serum (d > I.215 g/ml) were obtained from plasma of healthy individuals and prepared by c.cbntrifugation (Brown et al., 1974). Newborn and fetal calf lipoprotein-deficient serum at d > 1.215 g/ml (calf LPDS) were prepared bg centrifugation. 1251-labeled LDL was prepared as previously described (Goldstein et al., 1976). Reconstituted LDL was prepared by a previously described method (Krieger et al., 1978u). Th e endogenous apolar lipids of native LDL were removed by heptane extraction and replaced with either 25 hydroxycholestrryl oleate (designated r-[25-hydroxycholesteryl oleate]LDL: Krieger et a/.. 19783) or PMCA oleate (designated r-[PMCA oleate]LDL: Krieger et al., 1979). In each reconstitution, 1.9 mg of heptane-extracted LDL-protein was incubated wit,h either 209 ~1 of heptane containing 6 mg of 25.hydroxycholesteryl oleate or 200 ~1 of benzene containing 6 mg of PMCA oleate. The r-[25-hydroxycholesteryl oleate]LDL preparations contained approximately 93 mg of 25.hydroxycholesterol per mg of LDL-protein. The r-[PMCA oleate]LDL contained approximately 1.3 mg of PMCA oleate per mg of LDL-protein. lz5Tm labeled canine apo E-HDL, was a gift from Dr R,obert Mahley. 125T-labeled rabbit p-VLDL was prepared as previously described (Mahley et al., 1980). (c) Cell culture Chinese hamster ovary cells (CHO-Kl cells) were obtained from the American Typ (‘ulture Collection, Rockville, Md (cat no. CCL61). Cells were grown in plastic dishes and flasks (Falcon) at 37°C in an atmosphere of 50/6 CO,. Stock cultures were grown in medium A (Ham’s F-12 medium containing penicillin (100 U/ml), streptomycin (100 pg/ml), and 2 mMglutamine) supplemented with 5oj, (v / v ) newborn calf serum. Where indicated, the newborn calf serum was replaced with 3%, li”/&, or lOoi, calf LPDS. For cloning the cells by dilution plating, portions (1 ml) of medium A containing 100/b fetal calf serum and an average of 6.5 cell/ml were added to each well of a 24.well culture dish (Linbro or Falcon). All biochemical assays were performed using a standard format. Cells from stock flasks were dissociated with trypsin/EDTA solution. On day 0. 1 x lo5 cells were seeded into each 60.mm Petri dish in 3 ml of medium A containing 5% calf LPDS. On day 2, the medium was replaced with 2 ml of medium A containing 5 or lo?,& calf LPDS. All experiments were initiat,ed on day 3. For fluoresceme photomicroscopy, cells were seeded into dishes that contained glass coverslips and grown as described above. On day 3, cells received medium A containing 34, caalf LPDS and 10 pg protein/ml of r-[PMCA oleate]LDL. Aft,er incubation at, 37°C for 25 h.

170

M. KKIEGEH.

M. S. BROWN

ANI) .I. L. GOLDSTEIN

the cells were washed (Krieger et al., 1979) and fixed with 3% paraformaldehyde in @2 Msodium phosphate (pH 7.3) for 10 min at room temperature. The coverslips were removed from the dishes, washed with water, mounted with glycerol on glass slides, and viewed in the epifluorescence and phase contrast modes of a Zeiss Photomicroscope III equipped with the following filters: exciter filter, 365/12 nm; chromatic beam splitter, 395 nm; barrier filter, 420 nm (Krieger rt al., 1979). (d) Mutagenesis and isolation

of mutant

clones

On day 0, CHO cells were plated into 250-ml flasks (3 x IO5 cells/flask) in medium A containing 5% calf LPDS. On day 1, the medium was changed to medium A containing 5% calf LPDS and @4 mg of EMS/ml. After 19 h at 37”C, the cells were washed 3 times with phosphate-buffered saline and were then grown in medium A containing 5% calf LPDS. After an expression period of 6 days, the mutagenized cells were subjected to a 2-step procedure. (i) Step I: 69owth in toxic reconstituted LDL For the experiment described in Table 2, a total of 10’ mutagenized CHO cells were distributed in 50 dishes (100 mm) at 2 x lo5 cells/dish in 6.5 ml of medium A containing 30/o calf LPDS and 1Opg protein/ml of r-[25-hydroxycholesteryl oleate]LDL. Every 3rd day thereafter for 18 days, the cells were washed with phosphate-buffered saline and refed with fresh selective medium. (i) Step 2: Uptake of jluorescent reconstituted LDL After the 18.day incubation in medium containing toxic reconstituted LDL, the cells were washed with phosphate-buffered saline and refed with medium A containing 3% calf LPDS and 5 rg protein/ml of r-[PMCA oleate]LDL. After incubation for 2 days, the dishes were examined in situ with a Leitz inverted fluorescence microscope equipped with the following filters: exciter filter, 340-380 nm ; chromatic beam splitter, 400 nm; barrier filter, 430 nm. In t’he 50 dishes, a total of 86 well-defined colonies ( > 200 cells/colony) were observed, most of which were either not fluorescent or only slightly fluorescent. Fifty-three non-fluorescent colonies were isolated with cloning cylinders and transferred to 60-mm dishes containing medium A and 10% fetal calf serum. The cells were allowed to attach overnight, after which they were washed once with phosphate-buffered saline and refed with medium A containing 30/6 calf LPDS and 10 rg protein/ml of r-[PMCA oleate]LDL. Two days later, the isolates were examined in the fluorescence microscope. There was no detectable dye uptake in 20 of the isolates, while the remaining 33 isolates expressed very low to normal uptake. The 20 negative isolates were removed from the dishes with trypsin/EDTA solution and clones were created by dilution plating in the absence of r-[25-hydroxycholesteryl oleate]LDL, as described above. Fourteen of the 20 isolates yielded viable clones that were transferred to Petri dishes and retested by fluorescence microscopy for their ability to bind and internalize r-[PMCA oleate]LDL. Eleven of the 14 clones did not internalize detectable amounts of the fluorescent reconstituted LDL; these clones were maintained in medium A containing 5oj, calf LPDS and were subjected to biochemical characterization as described in Results (see Table 2, group A clones). These clones were each derived from a different dish in the original selection step. (e) =1says The rate of internalization and proteolytic degradation of human [ ’ “I]LDL by intact cells at 37°C was determined in medium B (medium A containing 107; human LPDS) as previously described (Goldstein & Brown, 1974). Degradation activity represents the celldependent rate of proteolysis and is expressed as the pg or ng of ‘251-labeled acid-soluble

LDL

KE(‘EPTOK

MI’TATIONS

IS

(‘HO

Ii1

(‘ELLS

(non-iodide) material released into the culture medium per mg of total cell protein. The binding to surface receptors at 37°C was measured by the dextran amount of [ lz51]LDL sulfatcb release technique (Goldstein et al., 1976). The total amount of ‘251-labeled human or rabbit fl-VLDL bound to the cell surface at 4°C was LDL. canine apo E-HDL,, determined as previously described (Goldstein & Brown, 1974). Binding activity is expressed RS np of [1251]lipoprot~in bound per mg of total cell protein. The inrorporat,ion of 1I“(‘Joleate into cholesteryl [14Cloleate by cell monolayers was measured as previously described (Goldstjein et al., 1974) and is expiessed as nmol of cholesteryl [‘4C]oleate formed/h per mg of total cell protein. The activity of HMG CoA reductase was measured in cell-fret, formed/min peer extracts as previously described, and is expressed as pmol of 1“C]mevalonate mg of detergent-solubilized protein (Brown et al.. 1974). The free and esterified cholesterol

vonttant of cells and the 25hydroxycholesteryl

oleate content of r-[25hydroxycholestvryl

oleatejLDL was determined by a previously described gas-liquid chromatographic method (Brown rl al., 1975) after lipid extraction of the cells or lipoprotein by the method of Folch p/ 01. (1957). The protein concentration of whole cells, cell extracts. and lipoproteins was tlet,errnined by the method of Lowry et al. (1951) with bovine serum alhumin as a standard. The uptake of I 3H Isucrose was measured by incubating cells at 37°C for varying times in at 7.73 x 106 disints/min per ml. 2 ml of medium A csontaining So0 calf LPDS and I ‘HIsucrose

.\ft’er incubation.

thr cells were washed 5 times with an albumin-containing

buffer ((:oldstrin

(1 (I/.. 1976), followed by a final wash in @15 M-NaU. The cells were harvested in 0.1.5 M-Sac’]. f)ellr+ed at, %(HH) revs/min, resuspended in 500 ~1 of 1096 (v/v) Trit)on X-100 for 15 min a.t room t.rmperatnrr. and counted in 10 ml of Aquasol (Kew England Nuclear Carp).

3. Results (a) Isolation

of wsistant

cl0ne.s

Our approach for isolating (“HO cells bearing mutations in surface receptors is based on the methodology that has been used successfully by a number of investigators for the induction and isolat,ion of drug-resistant mutations in mammalian cells (for reviews, see Siminovit,ch. 1976: Caskey & Kruh, 1979: Lewin. 1980). The details of the mutagenesis and selection procedure are given in Materials and Methods. After mutagenesis with ethylmethane sulfonate, t)he cells were allo\\-ed to recover and exJ)ress altered phenotypes. Thereafter. the cells were grown in lipoprotein-deficient serum in the presence of LDL reconstituted with 25. is designated hydroxycholesteryl which r-[2&hydroxycholesteryl oleate, oleate]LDL. This toxic reconstituted LDL is internalized by cells expressing the LJ)L receptor pat’hway. and the 2Shydroxycholesteryl oleate is hydrolyzed within Iysosomes (Krieger et a,l., J978h). The liberated 25hydroxycholesterol suppresses cbndogenous cholesterol synthesis at the level of HMG (‘0~4 reductase. The cells then become cholesterol-deficient, and die. Mutant human fibroblasts that) lack LDL receptors cannot take up the r-[25hydroxycholester;vl oleate]LDL and are not killed by this particle (Krieger et al.. J978b). In theory, a cell resistant to the killing effects of r-[25hydroxycholesteryl oleate]LDL could possess a defect at’ any one of the following st’eps in the LDL receptor pathway: binding, internalization. deliver) to lysosotnes. and lgsosomal hydrolysis of the reconstitut,ed LDL to yield t,he toxic 2.5hvdroxvcholesterol. In addition, the resistance could be a consequence of a resistance t,o t)he 25hydroxycholesterol itself. To eliminate the latter unwanted cells. the surviving colonies were subjected to an identification step in which they were incubat~ed in medium containing LDL reconstituted w&h a fluorescent cholesteryl

17%

M. KRIEGEK.

M. S. BRO\VN

AND

.I. I,. GOLDSTEIS

oleate derivative, which is designated r-[PMCA oleate]LDL. This reconstituted fluorescent LDL can accumulate in cells only if the cells express a functional LDL receptor pathway (Krieger rt al., 1979). To determine whether the clones isolated by the above procedure arose as a result of mutagenesis, we carried out an experiment in which parental (“HO cells were treated with or without EMS prior to selection (Table 1). When 3 x 106 nonmutagenized cells were tested, only one large colony ( > 200 cells/colony) survived the first step of the selection procedure. Seven small colonies (-30 to 200 cells/colony) were also seen in the dishes. When tested in the second step, all of these colonies, both large and small, internalized and accumulated the fluorescent r-[PMVA oleate]LDL, indicating the presence of a normal LDL recept’or pathway. Thus, without mutagenesis, the frequency of mutations affecting the LDL receptor pathway was less than 1 in 3 x lo6 (i.e. less than 3.3 x IO-‘/cell). In contrast. when the parental cells had been mutagenized, 20 large colonies and 70 small colonies survived the first selection step. In a random sample, six out’ of six large colonies examined, and two out of 16 small colonies, failed to int,ernalize and accumulate the fluorescent reconstituted LDL. Thus, the calculated frequency of mutations was 6.6 x 10m6 per cell for large colonies and 2.8~ lOA for small colonies (t,otal frequency, - I x 10V5). The frequency of mutations could also be calrulat’ed from a Poisson analysis of the distribution of surviving colonies. Based on the observed number of dishes containing no colonies, the calculated mutation frequency for large colonies after EMS treatment was 5.5 x 10Y6 mutat,ions per cell. a number t)hat was similar to the observed value of 6.6 x IV6 (see footnote 1. Table 1). Eleren mutant clones were isolated by the t’wo-step procedure in the experiment, described in Materials and Methods. These clones (group A clones) were each isolated from a separate Pet’ri dish. The clones were tested for t,heir abilit’y t’o metabolize [125 I]LDL (Table 2). The amount of surface-bound 1125TILDL and the degraded by the cells were measured. The high affinity amount of [ ‘*‘I]LDL binding values for the group A clank ranged from undetectable ( < 5 ng/mg prot,ein) t,o 250, cells. In each of the group A clones, the ’ 0 of the values in the parental defectire binding was associated with a proportional decrease in [“‘I JLDL degradation, the values ranging from 1‘& to 32qo of normal. Xo clone was observed in which degradation was reduced to a greater degree than multl be explained by the decrease in binding. and Four of the group A clones (17-2, 40-2, 1 l-l and 14-l ) were subcloned maintained in the absence of r-[25hydroxycholestergl oleate]LDL and are designat,ed 17.2a, 40-2~. II-la and 14.la. respectively. The 11-la and 14-la subclones were then re-cloned. again in the absence of r-[25hydroxycholesteryl oleate]LDL. and these clones are designated 11-la1 and 14.lal. respectively. These group .L\ subclones were tested for their ability to metabolize [‘*‘I]LDL (Table 2). The amounts of [ “‘1 ] LDL bound and degraded bv the subclones were similar t’o t’he values of the original clones. On the basis of the measured growth rates of the 11-la1 and 14-la1 cells (see below), these cells have undergone more than 200 population doublings in the absence of r-[25-hydroxycholesteryl oleatelLDL and have been subcloned twice. Throughout this time, they have shown no alteration in their receptor-deficient phenotypes. Under conditions of logarithmic growth. the

f,l)f>

ftE(‘EPTOK.

MI‘TATIONS

TABLE Isolation

of

r-r25-h,ynrox?/,/ch,olPskryl

IS

(‘HO

(‘ELLS

17:j

1

CHO cells resistant to : dqwadmw on mrrtagpn

oZeate]LDL

‘f’reatment

I-‘aIlmlc4c~r

Total number f)f c*effs f)fatetl No. of’ surviving rolonies resistant to r-/25 h~tfrox~~hofrstrrvl ofeate]LDf,: large (,ckmirs t small ~~olonirs E’rac.tion of’ examined colonies that f’aifed to internalize r-lPM(‘A ofcate\LDf,: large c~olrmies small I0lonir~ fktimated frequen(‘y of mutations f,Df, rec~rptor f,athwnj large (.ofonirs small tx)fonies total ~~ofonir~

No EMS

+ EMS

3 x IO6

3 x I oh

1 7

201 in

O/f o/7

G,/ti L’/lli

< 3.3 x I 0 - ’ <3.3 x lo-’ <3.3 x lo-’

6.6 x 10-y “.Xx 1or6 -I x lWS

affecting

(blls were treated with or without EMS as desc*ribed in Materials and Methods. after a retavery ancl repression period of 5 days, each set of cells was plated into 15 dishes at 2 x 105 c.efls/dish in medium A (9ntaininp :~‘I,, calf f,Pf)S and 20 pg protein/ml of r-1 P5-hvtiroxpcholester~f oleateJLDL. Fresh medium was added on days 4 and 8. On day 16: the dishes were examined microscopically. In the none mutagen&l offs. a tot,af of 1 large colony and 7 small colonies were seen. The dishes containing these (&)nies were incubated for 48 h in medmm A containing 39,0 calf LPDS and 10 rg protein/ml of r-1 PMC’A oleatrlLDfJ. All ofthese colonies, large and small, became fluorescent. These and the remaining ilishrs were then fisetf with methanol and stained with Cl’:, (w/v) ervstaf violet. ,Ir’o additional colonies were ~letr(~trtl. In the mutayenized cells. 20 large c*ofonies and 70 small c,olonies were detec*tetl tni~~ro~c~opi(~xffv. Three dishes containing a representative sample of 6 large c.ofonies and 16 small (&mics were kubatetf with r-IPMC’A ofeate]LDL as described above. All 6 of the large colonies. but only 2 of the Iti small caofonies, failed to show ffuores~~ent~e. All of‘ the dishes were then st,ained with ,yst,al violet as above and the total number of colonies was counted. An estimate of the t’requency of’ mutat,ions was calculated by dividing the number of surviving colonies that failed to internalize r-1 PMC’A ~~IeateJLf~L hv the total number ofceffs used for the selection. A similar value can he calcufatpd from a Poisson analysis of the tfistribution of surviving colonies (see footnote 1). t fCac,h faryr c~ofony c*ontained more than 100 cells. Each small colony contained approximately 30 to “00 IY~IIS. $ ‘f’hr (listribution of large colonies among the 15 replicate dishes of cells treated with EMS was as lidlm+.. Sumber of large csofonies/dish 0 1 2 3 4 >4

Sumber of dishes -5 4 3 2 I 0

Hasetl on the observed proportion of dishes containing no large colonies (5/15.I: = C33). the frequent? of induced mutations was calcufat,ed to be 5.5 x 10m6 mutations/cell. This c.alculation was made b> Poisson analvsis using the formula m = (-In (/‘,)/N). where m = mutation frequency antf N = number of ~11s pfat&dish (f,ewin. 1980).

174

M. KHIEGEK,

M. S. BKGM’S TAHLE

AS11 .I. I,. GOLllS’l’EIN 2

Metabolism of [ ‘251]LDIJ at 37°C its clonrs arbd subelorws oj OH0 cells resistant to r-[25-hydroxycholesteryl oleatv ]LIlL Metabolism

of j ‘251]LI)L

(:ell designat ion

High aftinit~ binding

High affinity tlegratlation

LDL rrc~eptoi aetivityt

Protein (,ontent

ndw 41 (“1.-Cl):

ng/rnp

0o control

&dish

Parental (‘HO (aells (iroup A--clones Ii-2 40-Z A4-2 38-2 11-l 14-l l-l D-P 121 421 Hi- 1

<5 <5 <5 <5 <5 < .5 <5 <5 ti 10 10

“30 (15tk3OO):

“2 I0 3(i 34 38

1 1 2 2 3

84

4

145 175 IS0 202

9 9 13 1x

493

:2 3

180 120 240 200 220 180 270

180 170

100 140

/I

17.“a 40.1(X

11.la1 14.la 14.la1

475-7-l 475-11-l

100

$

Group A-subrlones

Group B--clones 475-13-l

1846 (llW%9OO)~

<5 <5 <5

1” 27 49

3 2 2

230

<5

M

4

“10

< 5

!I4

9

I60

<5

60 til 102

2 2 4

230

190 180

‘1 <5 <5

210 Ifi0

On day 3 of’ cell growth, eac*h monolayer was incubated with 2 ml of medium B containing 15 pg protein/ml of [ ‘*‘I]LDL (140 to 244 cts/min per ng protein) in the presence or absence of 400 pg protein/ ml of unlabeled LDL. After incubation for 5 h at 37”C, the amount of [ “‘I]LDL degradation products excreted into the medium, the amount of surface-bound ] ‘LSI]LDL. and the cellular content of protein were determined as described in Materials and Methods. The data for binding and degradation are expressed as the high affinity values. which represent the difference between the values observed in the absence and preseme of unlabeled LDL. The values for the parental CHO cells represent the mean and range for 12 separate experiments. each determined in triplicate. Eat-h value for a resistant clone or subclone represents the average of triplicate incubations carried out in one experiment. t The ““,, control” value for LDL reeept,or activity represents the amount of high affinity [ ‘2”I]l~Dl~ degradation in the resistant clones relative to that in the parental CHG cells. as measured in the same experiment. 1 Itange of values from 12 experiments. 5 The mutagenesis, .seleetion, and cloning of the group A clones were carried out in the same experiment as described in Materials and Met,hods. Each clone was obtained from a different. dish. ]] Group .q subclones 17-2a, 40-2~ and 14.la were derived from clones 40.2, 17-2 and 14-1, rrsprc+ivrly. t:roup A subclones 14.la1 and 1 llal were cloned from 14.la and from a sub&me of 1 l-l. 11-la, respectively (see the text). 7 Group B clones were isolated in a separate mutagenesis experiment. The protocol was similar to that used to isolate group A clones except that the concentration of r-[25-hydroxycholesteryl oleat~e]l,l)l, was 20 ~g protein/ml of medium rather t,han IO pg protein/ml. Each of the group B clones was obt,ainetl from a different dish.

LDL

KE(‘EPTOK

MI”l’ATIOKS

IN

(‘HO

(‘EI,LS

I73

I)arental (‘HO. 14.la and 11-la cells had doubling times of 16. 19 and 23 hours. respectively. In another experiment. mutagenized CHO cells were selected by a similar twost,ep procedure, except that the concentration of r-[25-hydroxycholest,eryl oleate]LDL used in the first step was doubled to 20 pg protein/ml (group B clones in Table 2). As was the case with the group 4 clones, the values for [ 1251]LDL binding and degradation in the group B clones were much lower than those of the parental (‘HO cells (2 t’o -C”& of parental CHO activity). The results of the analyses in Table 2 suggest that all of the clones isolated by the two-step procedure were defective in the cell surface binding of LDL. This defect in I)inding presumably accounts for their ability to grow in the presence of the toxic, reconstituted LDL and their inability to internalize and accumulate the fluorescent reconstituted LDL. The mutant clones and subclones that expressed less tha,n 5”;) of wild-type LDL receptor activity were analyzed further to characterize the nature of their defects. Clones 11-l and 14-1 and their subclones were most thoroughly studied. Figure 1 shows the effect of r-[25-hydroxycholesteryl oleate]LDL on the relative plating efficiency of parental CHO cells and mutant clones 14-la and 11-la seeded at low density (250 tells/60-mm dish) in 3’7; calf LPDS. Colony formation by the parental CHO cells was inhibited by 507; at a concentration of 1 pg protein/ml of the r-]25hydroxycholesteryl oleate]LDL. In contrast, 14la cells (Fig. 1(a)) and I1 1a, cells (Fig. 1(b)) were resistant to the reconstituted LDL at concentrations as high as 1OOpg protein/ml, which is equivalent to a 25-hydroxycholesterol 125

I

\

0

\ 0

/

’ 0.A

I 1

0

‘0-q-o-T 10

x too 7

r-[25-Hydmxycholesteryl

, 04

-y-.-o 4

10

100

oleote] LDL (pg protein /ml)

NC:. I Relative plating efticiency of parental and mutant CHO cells grown in the presence of’ r-115. h~drc~,ay~holester~l oleate]LDL. On day 0, 250 cells were &seededinto no-mm dishes in 4 ml of medium A Iontammg W, calf LPDS and the indicated amount of r-[P5-hytlroxycholesteryl oleate]LDL. On day 7. the monolayers were washed. fixed with methanol and stained with O,l”/, (w/v) crystal violet, and the number of colonies per dish was counted. Each datum point represents the average of duplicate ~leterminntions expressed as the percentage of the number of colonies in control dishes. The IOO”,, control values were as follows: (a) 165 and 97 colonies/dish for parental (0) and 14-111 (0) cells, respectively : and (b) 172 and 148 colonies/dish for parental (0) and 11-la (A) cells. respectively. The plating efficiency observed when the cells were incubated in the presence of 10~~ of “5. h~drosvc~holeaterol~ml added to the culture medium in ethanol was zero for all 3 strains and is indicated by an k in (I)). 7

176

M.

KHIEGER.

M.

S. BRO\Z’N

AND

.I.

I,.

GOLDSTEIS

concentration of 30pg/ml. The addition of 10 pg of unesterified 25-hydroxycholesterol per ml (added to the cells in ethanol) completely prevented colony formation by the II-la and 14-la cells as well as by the parental (‘HO cells (Kg. l(b), X). indicating that the resistance of these mutant cells was not due to a resistance to 25hydroxycholesterol itself, but rat,her to an inabilit’y of these cells to take up the 25-hydroxycholesterol when it, was in LDL. Chromosomal analysis, performed by Dr V. Dev, showed no differences in the karyotypes for the 11-l a, 14-l a, 17.2a and 40-2~ cells, as compared to the karyotype of the parental CHO cells.

FIG. 2. Visualization of the uptake of fluorescent r-[PMCA oleate]LDi (C.1)). and 11-l (E,F) cells. On day 3 of cell growth, each monolayer containing 3”, calf LPDS and 10 pg protein/ml of r-[PMCA oleate]LDL. 37°C. the cells were washed and fixed, the coverslips were mounted for fluorescence (B,D.F) mivrosvopy. and photographs were made as desrribed Magnification. 150 x

by parental CHO (A,B). 14-l received 2 ml of medium A After incubation for 25 h at phase contrast (A,C.E) and in Materials and Methods.

LDL

RECEPTOR

MUTATIOXS

(11) Characterization internalization,

IN

(‘HO

(‘ELLS

177

of defects in LDI, hindkg, a,nd degrada.tiow

Figure 2 illustrates the dramatic difference in appearance of the parental (‘HO and mutant cells after incubation for 25 hours in the presence of r-[PMCA olrat,eILDL. Panels A. C and E show phase contrast micrographs of the parental ( ‘HO, l-i- 1 and 1 l-1 cells, respectively. Fluorescence micrographs of the same fields are shown in panels B. D and F, respectively. While each of the parent,al CHO cells intjr>rnalized and accumulated substantial amounts of the fluorescent, dye. there \vas virtually no uptake of the dye by the mutant cells. To determine whether the reduction in LDL binding in the apparently receptornegative mutant clones was due to an alteration in affinity of the receptor. MY pttrformed [ ‘251]LDL sat,uration curves, using the rate of [ ‘251]LDL degradation as a measure of receptor binding (Fig. 3). None of the mut,ant clones showed evidence at lipoprotein concent’rations as for a saturable degradat’ion process for [125 I]LDL high as 100 pg protein/ml. Similar results were obtained for clones 17-2, 475-7-l. 17.5-13-l and 475-11-l (data not shown). 5

Parental

CHO Cells

[ 1251]L!JL

(pg protein/ml)

FIG. 3. Saturation kinet,ics for the degradation of I”’ I]LDL by monolagers of parental and mutant (‘HO cells at 37°C’. On day 3 of cell growt,h. each monolayer received 2 ml of medium B containing the indicated amount of human [ lz51 ] LDL (81 cts/min per ng protein). After incubation for 5 h at 37°C. t,he amounts of 1lz51 ] LDL degradation products excreted into the medium were determined as described in Materials and Methods. The values for the parental CHO cells (0) represent the mean_fs.D. from 4 separate experiments. The values for each mutant clone represent single incubations. The mutant clones examined were: 14-l (0): 11-l (A); A4-2 (0); 40-2 (m); and 38-2 (A),

To compare directly the LDL receptor binding activity of the parental CHO cells and one of the mutant clones (14-l), monolayers were incubated at 4°C with three lipoproteins that bind to the LDL receptor of parental CHO cells with different affinities : human [ 125I]LDL (Kd = 7 pg protein/ml) : rabbit [ 1251]p-VLDL (Kd = 0.04 pg protein/ml): and canine apo E-HDL, (Kd = 0.1 pg protein/ml) (Fig. 4). The 14-1 cells showed no significant high affinity binding of any of these ligands. A similar lack of high affinity binding of [1251]LDL, [1251]fl-VLDL 01 [ ‘251]apo E-HDL, at 4°C has been observed with clones 17-2,40-2c, A4-2 and 1 l-la. In other binding experiments at 4”C, we observed that the affinity for canine

M. KRIEGEK.

M. 8. BROWN

(a)

AND

-

.I. 1,. GOLDSTEIN

(b) -

5

0

0.5

IO 0

(c’

05

i-0 0

I.0

pg protein/ml Fro. 4. Saturation kinetics for the binding of I”‘I]LDL (a), ( ‘Z51]/z-Vl~DL (b). and [ ‘Z51]apo E-HDL, (c), to parental CHO (0) and 14-l (0) cells at 4°C. On day 3 of cell growth. each dish was chilled at 4°C for 30 min. after which was added 15 ml of ice-cold medium B containing the indicated amount of human [ lz51]LDL (211 cts/min per ng protein). rabbit 11z51],%VLDL (648 cts/min per ng protein). or (890 cts/min per ng protein). After incubation for 2 h at 4°C. the amount of canine 1lz51 ] a po E-HDL was determined as described in Materials and Methods. Each datum point cell-bound [ lz51 ] lipoprotiin represents the average of duplicate incubations. Each panel represents a separate experiment.

(al

,n, -0

2

4

6

0

Incubation

0

2

4

6

8

time (h)

(b) by parental CHO (O), 14.la Flu. 5. Time-course of the uptake of [‘HIsucrose (a) and 1“‘I]LDL (O), and 1 l-la (A) cells. On day 3 of cell growth, each monolayer received 2 ml of medium A containing 5Y,, calf LPDS and either 7.73 x lo6 disints/min per ml of 13H]sucrose (11.2 Ci/mmol) or 15 pg protein/ml of-1 ‘2SI]LDL (83 cts/min per ng protein). The dishes were then incubated at 37°C for the indicated times. (a) The amount of cell-associated [ “HIsucrose was measured as described in Materials and Methods. (b) The total cellular content of [ lz51]LDL and the amount of [ lz51]LDL degraded by the cells were measured as described in Materials and Methods, and the sum of these 2 values is plotted. Each datum point represents the average of duplicate incubations.

LI)L

I~ECEPTOK

MVTATIOSS

IN

(‘HO

179

(‘ELLS

1“51]apo E-HDL, in the 12-l cells, a clone that had reduced but detectable LDL receptor activity at 37°C (Table 2), was similar (0.12 pg protein/ml) to that’ of the parental CHO veils (0+13 pg protein/ml). These data suggest that the residual LDL receptor activit:\: in the 12-1 cells is not due to a mutation altering the binding aftin&\- of the receptjor. Whereas t)hr l&la and 1 l-la cells were unable to internalize and degrade 1‘151 ILDL with high affinity (Fig. 5(b)). both strains had a normal ability to take up 13H Jsucrose. a molecule that enters cells by bulk phase pinocytosis (Fig. 5(a)). From the rate of 13H js!lcrose uptake. we calculated that the parental (“HO cctts ingested approximately 0.15 ~1 of medium per mg of cell protein per hour. white the ~~~~mparable values in 11-l a and 11-l a cells were 027 and 0.12 pt. respectively. ((2) Swondary

allerations

in choksterol

tttrfoholisttt

In (‘HO cells and human fibrobtasts, cholesterol metabolism is regulat,ed b,~ I,l)t,-c~holrst,et.ot delivered vin the LDL receptor (Goldstein et al.. 1979b). LDLc~t~otesterot suppresses the activity of HMG CoA reductase. thereby decreasing endogenous cholesterol synthesis, and stimulates the activity of a+ C’oA : vhotesteryl acgltransferase. thereby increasing the cellular contint of esterified cholesterol. Inasmuch as LDL-cholesterol cannot be delivered to t,he mut)a,nt (‘HO cells t,hrough the LDL receptor pathway, their cholesterol metabolism should not, be regulated normally by LDL. Figure 6 shows t,he results of an experiment, in which parental and 14-l cells were incubated for 5 hours with varying amounts of either native LDL (Fig. 6(a)) or 18-l25.tl?ldroxyc,hotesteryl oteate]LDL (Fig. 6(b)), after which HMG (‘oA reductase

0

20

40

60

80

(a)

_

100

0

pg

(b)

2

4

6

8

10

protein/ml

kc:. 6. Suppression of HMC CoA reductase ac%ivity in parental CHO (0) and 14-l (0) cells hy LDL (a) and r-/25-hvdrolv~holester~i oleate]LDL (b). On day 3 of cell growth. each monolayer received 2 ml of medium A conkining lo”,, calf LPDS and the indicated amounts of native LDL (a) or r-125h?droxvc,holester~I oleate]LDL (b). After incubation for 5 h at 37”C, the cells were harvested and the HM(: CoA reductase activity was measured as described in Materials and Methods. Each datum point represents the results from duplicate (CHO) or single (14-l) incubations expressed as the percentage of vneyme act,irity in control incubations. The 100~~ control values were 624 and 647 pmol min-’ mg protk ’ for (‘HO and 14-l cells, respectively.

M. KKIEGEK,

180

-0

25

50

M. S. BKOM’N

75

100” 0

AND

5

J. L. GOLDSTEIN

10

15

20

25

”

LDL (pg protein/ml) lk. 7. Ability of LDL to stimulate the formation of cholesteryl 1“C]oleate (a) and the accumulation of unesterified (b) and esterified (c) cholesterol in parental CHO (0) and 14.la (0) (*ells. (a) On day 3 of cell growth. each monolayer received 2 ml of medium A containing 104, calf LPDS and the indicated amount of LDL. After incubation for 5 h at 37°C. the cells were pulse-labeled for 2 h with @l mM) “C]oleate-a1bumin (5580 cts/min per nmol). The cellular content of cholesteryl 1‘%Joleate was measured as described in Materials and Methods. Each datum point represents the results from a single incubation. (b) and (v) On day 3 of cell growth, each monolayer received 2 ml of medium .4 c-ontaining loo,, calf LPDS and the indicated amount of LDL. After incubation at 37°C for 24 h, the cells were harvested and the mass of unesterified cholesterol (b) and esterified cholesterol (e) were measured as described in Materials and Methods. Each datum point represents the average of duplicate determinations on samples pooled from 2 dishes,

was measured. Both of these lipoproteins suppressed reductase activity in the parental CHO cells. As previously found for human fibroblasts (Krieger et al., 107%). the reconstituted LDL carrying 25hydroxycholesteryl oleate was significantly more effective at suppressing reductase activity than was native LDL. In contrast, reductase activity of the 14-1 cells was not significantly suppressed by either lipoprotein. In other experiments, not shown. we found that the reductase activity of clones 17-2, 11-la, 40-k and A4-2 was also resistant to suppression by native LDL at concentrations as high as 100 or 2OOpg protein/ml. Figure 7(a) shows an experiment in which increasing amounts of LDL were incubated with parental CHO cells and 14-la cells, after which the ability of these cells to incorporate [14C]oleate into cellular cholesteryl [ 14CJoleate was measured. With increasing amounts of LDL added to the incubation medium, the parental CHO cells exhibited a marked stimulation of cholesterol esterificat’ion (>25-fold), whereas the stimulation by LDL in the 14-la cells was less than twofold. Similar results were obtained with mutant clones 11-la, 17-2,38-2,475-11-l, 40-2~ and A4-2 (data not shown). In another experiment, the cellular content of unesterified cholesterol (Fig. 7(b)) and esterified cholesterol (Fig. 7(c)) in parental CHO and 14. 1a cells was measured as a function of the amount of LDL added to the incubation medium. When the cells had been grown in the absence of LDL, the cellular content activity

LI)L

KE(‘EPTOK

None

MUTATIONS

IS

Compoctin + mevolonate

CHO

(‘ELLS

IS1

Compactin + mevalonote + LDL

Parental CHO cd Is

14-I

I I-la

PI<:. 8. Stained c&lnies of parental and mutant (“HO cells under conditions in which growth is tlependent on the prescnc’e of LUL receptors. On day 0. CHO, 14-l and 11-1~. cxellx were seeded at lO.000 ~~rlls/lWmm Petri dish in 8 ml of medium A containing 3% calf LPDS and one of the following arlditions: none: 40 PM-vompavtin and 250 PM-sodium Dr.-mevalonste: or 40 ,uM-compactin. 250 PMsodium nl,-mevalonate. and 3 pg protein/ml of LI)L. On days 3. 5 and 8. the c,ellx were refed with the indicated medium. ant] on day 10 the tell monolayers were washed. fixed and stained with Ol”,, (w,!r) (.rystHI violet.

of unesterified and ester&d cholesterol was identical in both cell strains. The unrstrrified cholesterol content did not increase in either strain when increasing amounts of LDL were added to the incubation medium (Fig. 7(b)). In contrast. the contentj of estrritied cholesterol in the parental CHO cells increased markedly with increasing amounts of LDL in the incubation medium, whereas LDL had no effect on the cellular content of esterified cholesterol in the ll-la cells (Fig. 7(c)). If the mutant (‘HO clones cannot obtain cholesterol from LDL. then these cells should not be able to survive under conditions in which cell growth is dependent on the delivery of LDL-cholesterol via LDL receptors. To test this prediction. we grew parental (‘HO. 11-l and 11 -I cells under conditions in which the cells’ HMO (‘0~4 reductase activity was blocked by compactin. a potent competitive inhibitor of the reductase (Goldstein et al.. 1979b). A low concentration of mevalonate was also a,dded t,o the medium so that the cells could synthesize the small amounts of mevalonate-derived products other than cholesterol t,hat are necessary for cell growth. Previous studies in human fibroblasts and (‘HO cells have shown that

182

M. KKIEGEK.

M. Si. HHO12’9

ASI1

.I. L. 00Lt)STEIN

under these conditions cells do not, have sufficient mevalonate for cholesterol synthesis. and they will therefore not survive unless t,hey can a,cquire cholesterol from LDL (Brown & Goldstein. 1980). When IO.000 parental (‘HO cells were seeded in t,he presence of lipoprotein-deficient serum. t,hey grew int’o healthy colonies when no drugs were added to the medium (Fig. 8. left column). In the presence of 40 palcompactin and 250 PM-mevalonate. the cells did not grow because they could not, make enough cholesterol for growth and they had no exogenous source of cholesterol (Fig. 8. center column). When LDL was added t,o t,hr medium containing compactin and mevalonate, the parental cells grew into caolonies (Fig. 8. right. column). In contrast. LDL could not rest’ore the growth of the t’wo mutjant clones 11-l and 11-la. which lack LDL receptors.

4. Discussion This paper reports the isolation of mutant (YHO cells with defects affecting the expression of LDL receptor activity. The mutations were induced with EMS. and the mutants were isolated using a two-step technique. In the first step, toxic reconstituted LDL, r-[25-hydroxpcholestervl oleate]LDL, was used t’o kill most of the cells that expressed normal LDL receptor activity. In the second step, a fluorescent reconstituted LDL, r-[PMCA oleate]LDL. was used to identify colonies of LDL receptor-deficient cells from among the few colonies that survived the first selection step. The altered phenotypes of the recept’or-deficient cells are believed t’o arise as a result of mutation because: (1) the appearance of such cells required mutagenesis with EMS: (2) the altered phenotypes were st,able when the cells were cloned, subcloned, and grown for extended periods under non-selective conditions ; and (3) when re-tested after growth for an extended period under non-selective conditions, the mutant (‘HO &rains were able to grow in t’he presence of a concentration of r-(2.5.hydroxycholesteryl oleate]LDL that was more than lO@fold above t’hat, which killed the parental cells and IO-fold above that used for the first selection step. The estimated frequency of induced mutat’ions in the LDL receptor pathway was approximately I x IO-’ per ~11. This is similar to the value of 2 x 10P5 observed for the induction of recessive emetine-resistant mutants with EMS in CHO cells (($upta & Riminovitch, 197X). All of the mutants isolated by the current method were found to have defects in the cell surface binding of LDL. We have not yet’ found an induced mutation in the (‘HO cells that is analogous to the natural mutation in the ,J.D. strain of human fibroblasts in which receptor binding is normal but internalization does not occur (Brown & Goldstein, 1979). If t,heg had occurred. such internalization mutations would have been detected by the current, protocol because the amount, of fluorescence accumulated in t’he presence of r-[PMCA oleate]LDL would have been markedly reduced. It is not yet known whether the mutant strains of (‘HO cells have defects in the structural gene for t’he LDL receptor. or whether the defects in these cells involve other genes whose products are necessary for t,he expression of the receptor on the cell surface. Such defects could involve proteins that participat,e in the regulation of receptor synthesis, post-synthetic glycosplation. targeting of the receptor to the

1,111, IIE~'E:PTOK

ML~TATIONS

IN ('HO C‘ELLS

1X3

cell surface. or recycling of the receptor. In preliminary studies, we have observed t,hat LDL receptor activity in one of the mutant strains, 11-1, can he restored when these cells are fused with cells from any of the other mutant strains listed in Table 2 unpublished observations). Thus, among LVright,, Brown & Goldstein, (Krieger. the mutants so far isolated, at least two complementation groups exist, implying that defects in at least two genes can create the receptor-deficient phenotype. In human tibroblasts t,he mutant alleles at the LDL receptor locus in patients w.ith familial hypercholesterolemia are expressed in a co-dominant fashion ((Goldstein & Brown, 1979). Fibroblasts from het’erozygous individuals express almlt XP,, of the normal number of receptors, whereas cells from receptor-negative homozygotrs express
M. KKIEGER,

184

M. S. BROM’N

A?17U .J. L. GOLDSTEIN

Debra Nobel and Jean Helgeson provided excellent technical assistance. This research was supported by a United States Public Health Services research grant (HL-20948) from the National Heart, Lung, and Blood Institute. One of the authors (M.K.) was the recipient of a U.S.P.H.S. postdoctoral fellowship from the National Heart, Lung, and Blood Institute. REFERENCES Attie, A. D., Pittman,

R. C. Br Steinberg, D. (1980). Proc. Ynt. =Icad. SC%.. C!.S.A. 77. 5923-

5927.

Basu, 8. K., Goldstein, J. L., Anderson, R. G. W. dz Brown. M. S. (1976). Proc. Sat. Acud. Sci., l~.S.A. 73, 3178-3182. Brown, M. S. & Goldstein, J. L. (1979). Proc. ,V’at. ;Icud. Sci., l!S.=l. 76, 3330-3337. Brown, M. S. & Goldstein, J. L. (1980). J. Lipid Res. 21, 505-517. Brown, M. S., Dana, S. E. & Goldstein, ,J. L. (1974). J. Biol. Chem. 249, 789-796. Brown, M. S., Faust, J. R. & Goldstein, J. L. (1975). J. Clin. Invest. 55, 783-793. Brown, M. S., Faust, J. R.. Goldstein. J. L., Kaneko. I. & Endo, A. (1978). J. Biol. Chem. 253, 1121~1128. Caskey, C. T. & Kruh, G. D. (1979). 012, 16, 1-9. Folch, tJ.. Lees, M. & Stanley, G. H. S. (1957). J. Biol. Chem. 226, 497-509. Goldstein, J. L. & Brown, M. S. (1974). J. Riol. Chem. 249, 5153-5162. Goldstein, J. L. & Brown, M. S. (1979). A nnu. Rev. Genet. 13: 259-289. Goldstein, J. L., Dana, S. E. & Brown, M. S. (1974). Proc. Xat. dcxzd. Ski.. l,T.S’.;I. 71. 42884292. Goldstein, J. L., Basu, S. K., Brunschede, G. YT. & Brown, M. S. (1976). (Jell, 7. 85-95. Goldstein, ,J. I~.. Anderson, R. G. W. 8: Brown, M. S. (1979a). Mature (London), 279, 679685. Goldstein, J. L., Helgeson, .J. A. S. & Brown, M. S. (19796). J. Biol. Chem. 254, 5403-5409. Gupta, R. 8. & Siminovitch, L. (1978). Somatic Cell Tenet. 4, 77-93. Krieger. M., Brown, M. S., Faust. J. R. & Goldstein, J. L. (1978u). J. Biol. Chem. 253,40934101. Krieger, M., Goldstein. *J. L. $ Brown, M. S. (19786). Proc. Nat. dead. Sci.. l:.S.A. 75,50X5056. Krieger, M., Smith, L. (‘., Anderson, R. G. W., Goldstein, J. L., Kao. Y. J., Pownall, H. ?J., Gotto, A. M. Jr & Brown, M. S. (1979). J. Suprumol. Struct. 10. 467-478. Lewin, B. (1980). Gene Expression. 2nd edit., pp. 142-188, John Wiley and Sons, New York. Lowry, 0. H., Rosebrough. N. ,J., Farr, A. L. 8: Randall, R. .J. (1951). J. BioZ. Chem,. 193, 26527.5. Mahley, R. W., Innerarity, T. L., Brown, M. S., Ho. Y. K. & Goldstein, ?J.L. (1980). J. Lipid Rex. 21, 97&980. Murray, (:. ,J. & Neville, D. M. Jr (1980). J. Rio/. Chem. 255, 11942-11948. Siminovitch, L. (1976). Cp/Z. 7, l-1 1. Weisgraber, K. H., Innerarity, T. L. & Mahleg. R. W. (1978). J. Hiol. Chum. 253,9053-9062.

Edited

by M. S. Rret.rcher