Isolation of an endocytic compartment from A431 cells using a density

modification procedure employing a receptor-specific monoclonal

antibody complexed with colloidal gold

JULIA BEARDMORE, KATHRYN E. HOWELL*. KAREN MILLER and COLIN R. HOPKINS

Biochemistn,- Department, Imperial College of Science and Technology, London SW7 2AZ, UK

•Present address: European Molecular Biology Laboratory, Postfach 10.2209, D-6900 Heidelberg, Federal Republic of Germany

Summary

Our objective was to isolate a prelysosomal com-partment involved in receptor-mediated endo-cytosis in human epidermoid carcinoma (A431)cells. The isolation protocol involves densitymodification of endosome elements in A431 cells,caused by the receptor-dependent binding andinternalization at 20 °C of colloidal gold-trans-ferrin receptor antibody (B3/25) particles. Theuse of 12sI-labelled gold-B3/25 provides a radio-active marker for the endosome compartment,the major peak being recovered at the bottom ofa continuous sucrose gradient at a density of1-23 g ml"1. Enzyme markers characteristicof other cytoplasmic compartments are presentonly in negligible amounts in this fraction and L-[35S]methionine-labelling of the cells indicatesapproximately a 200-fold enrichment of 12SI-labelled gold-B3/25 versus protein. Electron

microscopy of the endosome-rich fraction revealsthat we have isolated a highly purified populationof small gold-containing vesicles and tubulesfrom which the transfertin receptor can beimmunoprecipitated using the B3/25 antibody.Gel electrophoresis and fluorography of L-[35S]-methionine-labelled cells suggests that theseelements contain a characteristic profile ofapproximately 10 major proteins of which threeappear to be specifically enriched. In cellsincubated with [123I]transferrin, 12% of theligand sediments with the gold-labelled elements.We conclude, therefore, that the components wehave isolated play a role in the intracellularprocessing of the transferrin-transferrin receptorcomplexes.

Key words: A431 cells, colloidal gold, density shift,endosome isolation, transferrin receptors.

Introduction

A class of prelysosomal organelles that are acidic(pH5-6), and responsible for the uptake and process-ing of specific ligands and their receptors, has beendescribed in many cell types. These organelles havegenerally been termed endosomes because of their rolein endocytosis. It is becoming evident that they are ofprimary importance for the recycling, sorting andintracellular routing of many internalized proteins.Endosomes have heterogeneous morphology, appear-ing vacuolar and cisternal in shape, and having both aperipheral and a juxtanuclear distribution in many ofthe cell types studied (Helenius et al. 1983; Hopkins,1983a; Steinman et al. 1983).

In previous studies endosome-containing subcellularfractions have usually been isolated and identified by

Journal of Cell Science 87, 495-506 (1987)Printed in Great Britain © The Company of Biologists Limited 1987

virtue of their content of internalized radiolabelledligand. In these studies the buoyant density of thefraction containing endosomal elements has variedbetween 1-02 and l -Ugml" 1 depending on the gradi-ent material and subtraction of endosome elementsisolated. These fractions have also been shown to beenriched in certain cell surface receptors and to lackhigh activities of the enzyme markers for other cellmembrane compartments. Recent findings (Gallowayet al. 1983; Saemark et al. 1985; Tycko & Maxfield,1982; Van Renswoude et al. 1982) have also demon-strated the presence of an ATP-dependent protonpump in fractions containing endosome membranes,which may largely account for the acidic pH of theendosome content. The information so far available isthus consistent with a view of the endosome as adistinct cytoplasmic compartment, diverse in form and

495

function, and separate from the previously character-ized membrane boundaries of the plasma membraneand lysosomes.

Endosomes cannot be purified by their naturalbuoyant density alone because of close similarity withthe density of other cellular organelles. In such circum-stances selective density modification can be used toadvantage (Leighton et al. 1968; Trouet, 1964),specific examples being the purification of a low-density compartment involved in the uptake of galac-tose-exposing proteins in rat liver, reported by Quin-tart et al. (1984) and the separation of endocytic fromexocytic coated vesicles using a cholinesterase-me-diated density shift (Helmy et al. 1986).

In recent studies, the internalization and routing oftransferrin and the transferrin receptor in humanepidermoid carcinoma A431 cells has been described indetail using transferrin peroxidase (Tr-HRP) and atransferrin receptor-specific antibody (B3/25) com-plexed to colloidal gold (Hopkins & Trowbridge, 1983;Hopkins, 19836). Both transferrin and its receptorenter peripherally located endocytic compartmentsafter short incubations with labelled ligand at 37 °C.Similar endocytic compartments have been identifiedby Harding et al. (1983), in rat reticulocytes usingtransferrin-gold complexes, by Willingham et al.(1983), in human KB cells, and by Yamashiro et al.(1984) in CHO cells. It has been shown that thetransferrin-receptor complexes are continuously re-cycling back to the cell surface under these conditions(Harding et al. 1983; Hopkins & Trowbridge, 1983;Karin & Mintz, 1981; Klausner et al. 1983; Octave etal. 1981). During longer incubations (30min) andcontinuous exposure to transferrin or anti-transferrinreceptor antibody, the A431 cells transfer a proportionof the internalized ligand to a juxtanuclear compart-ment (Hopkins & Trowbridge, 1983), which is ident-ical to that which has been shown to contain epidermalgrowth factor (EGF) and EGF receptors in these cells(Miller et al. 1986). Similar incubations with eitherEGF-HRP, Tr-HRP and B3/25 gold at a temperatureof 20°C, heavily labels the juxtanuclear compartmentwithout significant labelling of lysosome-like elementsoccurring (Hopkins & Trowbridge, 1983; Miller et al.1986). Juxtanuclear concentrations of internalizedligands have been demonstrated in other cell types(Willingham et al. 1983, 1984; Yamashiro et al. 1984).

In the present study we have exploited the density ofgold colloid-transferrin receptor antibody complexesto isolate, by centrifugation, the endosomal compart-ments in which internalized ligand-receptor complexesbecome concentrated. This is possible because thedensity of colloidal gold complexes is sufficient tomodify the density of the membrane-bound compart-ments that contain them and also because previous

studies of intact cells have shown that, under appropri-ate conditions, these complexes can be internalized andconcentrated within the prelysosomal elements in-volved in the intracellular processing of cell surfacereceptors. In order to harvest receptor-rich com-ponents from both the peripheral and juxtanuclearregions of the endosome and exclude lysosomal el-ements, we have used continuous incubations withgold-antibody complexes at 20°C (Miller et al. 1986).

Materials and methods

Antisera, reagents and radiochemicalsThe B3/25 monoclonal antibody was a gift from Dr I.Trowbridge, Department of Cancer Biology, The SalkInstitute for Biological Studies, San Diego, CA 92138. It wasraised against the human haemopoietic cell line, K562, andhas been shown to be specific for the transferrin receptor(Trowbndge & Omary, 1981).

Mouse epidermal growth factor (EGF) was a gift from DrH. Gregory, I.C.I. Pharmaceuticals p ic , Alderley Park,Macclesfield, England.

Human transferrin (essentially iron-free), trypsin (EC3.4.21.4), soybean trypsin inhibitor type 1 and phenylmeth-ylsulphonyl fluoride (PMSF) were obtained from SigmaChemical Co. Ltd, London, as were the following enzymeassay reagents: p-nitrophenyl phosphate; cytochrome c typeIII from horse heart; /3-nicotinamide adenine dinucleotidereduced form (NADH); p-nitrophenyl-A'-acetyl-/3-D-glu-cosaminide; /J-glycerophosphate; adenosine; adenosine 5'-monophosphate (AMP) type III ; adenosine 5'-triphosphate(ATP);/>-iodonitrotetrazolium violet (INT), and fetuin typeIII. The terminal sialic acid and penultimate galactoseresidues were removed from fetuin by the method of Kim etal. (1971), to provide an acceptor for the enzyme galactosyltransf erase.

Na12SI (carrier-free) 1 5 m C i ^ " ' ; [2-3H]adenosine 5'-monophosphate (11-7 CimmoP1) and uridine diphospho-D-[6-3H]galactose (15-7Cimmol~') were purchased fromAmersham International, Amersham, UK.

Cell maintenance and incubation procedureA431 cells were grown in Dulbecco's modified minimumessential medium (DMEM) containing S % foetal calf serumand were plated out at 2X106 cells/9 cm Petri dish at least 3days before use. Confluent dishes of cells were washed free offoetal calf serum and incubated with 12sI-labelledgold-B3/25 or radiolabelled ligand in binding medium(DMEM, lOmM-Pipes, 100figml"1 bovine serum albumin,0-01 % Carbowax 20M, pH7-4), for 1 h at 20°C.

To follow the distribution of cellular protein and phospho-lipid during the isolation procedure, confluent dishes of cellswere either incubated with 50^Ci L-[3SS]methionine inmethionine-free medium or with 3 jtCi [met/iy/-3H]cholinechloride, for 18 h. Cells were washed to remove unincorpor-ated L-[3sS]methionine before incubation with gold-B3/25.The incorporated L-[35S]methionine was used to estimate theprotein distribution because colloidal gold interfered inconventional assays.

496 jf. Beardmore et al.

In some experiments cells were labelled for 1 h at 20 °Cwith gold-B3/25 followed by a brief wash at 4CC in bindingbuffer and a further 20-min incubation on ice with 5 ng/dish[12SI]EGF. Cells were lysed and fractionated as described inthe isolation procedure. The position of [ l i sI]EGF indicatesthe position of the plasma membrane on these gradients.[125I]EGF was added directly to control gradients to indicatethe position of free peptide.

Manufacture and handling of gold-B3/25 complexesGold particles (12 nm) were made according to the methoddescribed by Tolson et al. (1981), and stabilized with B3/25as described (Hopkins & Trowbridge, 1983). Before use, thegold complexes were washed by centrifugation in 001 %Carbowax 20M for ISmin at 25 000#, in a Beckman L8-S5ultracentrifuge (Beckman Instruments Inc., Spinco division,1117 California Avenue, Palo Alto, CA 93404). The com-plexes were resuspended in binding medium for incubationwith cells (approximately 100 jA of stock gold-B3/25 per9cm dish of cells).

lodinationTransferrin, B3/25 and EGF were iodinated according to themethod of Hunter & Greenwood (1962). Samples (50 fig,44^gor 2/ig, respectively) were iodinated using 0'5 mCi 12SI.Free 1Z5I was removed by column chromatography on BiogelP60 (transferrin and B3/25) or Biogel P6 (EGF) (Bio-RadLaboratories, Richmond, CA) in phosphate buffer, pH7-4.The specific activity of the radiolabelled proteins was7/xCi^g"1 for transferrin and B3/25 and 110/iCi^g"1 forEGF. The iodinated peptides were divided into aliquots andstored at — 20°C until used.

I25I was incorporated into the gold-B3/25 complexes byincubating 5 fid 125I with 0-5 ml gold-B3/25 for 1 h at roomtemperature. The I-labelled gold-B3/25 preparation waswashed twice by centrifugation at 25 000 £ for 15 min in0-01 % Carbowax 20M, and stored at 4°C.

Isolation protocolLysis. Following incubation with gold-B3/25, cells were

washed three times with ice-cold isotonic STEA buffer(0-25 M-sucrose, lOmM-triethanolamine, lOmM-acetic acid,1 mM-MgCl2, pH7'4) a variation of that described by Stahnet al. (1970). Cells were scraped off the dish in the minimumvolume of STEA buffer, using a rubber scraper, and lysed onice by pipetting with a 50— 250/il Finnpipette (LabsystemsOY, Pulttitie 9, 00810 Helsinki 81, Finland). As judged byphase-contrast microscopy, at least 80 % breakage of the cellswas achieved.

The homogenate was treated with 0-5/igml"1 trypsin for3 min at 37°C, before O-Sf/gml"1 soybean trypsin inhibitor(SBTI) was added and the homogenate was cooled on ice.Unbroken cells and DNA aggregates were removed bycentrifugation at 800g for lOmin at 4CC ( = nuclear pellet).The resulting 'post-nuclear supernatant' (PNS) was centri-fuged three more times as above, each time transferring thesupernatant to a clean tube. The final supernatant was againtreated with trypsin (0 -5 j igmr \ 3 min at 37°C) and SBTIas before, and PMSF was added to 1 mM.

Fractionation. Gradients were formed in Beckman 14 mlUltra-Clear centrifuge tubes (Beckman Instruments Inc.,

Spinco division, 1117 California Avenue, Palo Alto, CA94304) by first forming a 0-7 ml 4 % agar pellet (made in 52 %sucrose) at the bottom of each tube. A 0' lf imXl3mmdiameter Millipore filter (Millipore Corporation, Bedford,MA 01730) was placed on top of the agar pellet and a 12 mlcontinuous linear sucrose gradient (26 % to 52 % sucrose in10mM-tnethanolamine, lOmM-acetic acid, 1 mM-EDTA, pH7-4) formed. PNS supernatant (from approx. 12X106 cells)was layered on top of the sucrose gradient and then centri-fuged in a Beckman SW40 swinging bucket rotor at 200 000£for 18 h at 4°C in a Beckman L8-55 ultracentrifuge.

Gradients were fractionated from the bottom (0-5 cmabove the filter) into 1 ml fractions. The filter, agar and thesucrose solution just above the filter were each removedseparately for biochemical analysis or processing for electronmicroscopy. Cellular material was removed from the filter byimmersing it in 52% sucrose and gently pipetting or scrapingthe surface with a razor blade so that enzyme assays could beperformed on this fraction (fraction 1). The efficiency of thisprocedure was monitored by radioactivity. The radioactivecontent of the fractions was counted immediately, and withthe exception of galactosyl transferase activity, which wasassayed in fresh fractions only, the fractions were stored at—20°C for 1-3 days to await further analysis. The sucroseconcentration in fractions was measured at 25 °C using aBellingham and Stanley pocket refractometer, and convertedto sucrose density (gml"1).

Enzyme assaysMarker enzymes were assayed according to the followingreferences: galactosyl transferase (EC 2.4.1), using asialo andagalactosyl fetuin as acceptor (Howell et al. 1978); 5'nucleo-tidase (EC 3.1.3.5) (Stanley et al. 1980); succinate-INTreductase (INT, 2-(4-iodo-phenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (EC 1.399.1) (Pennington,1961); NADH-cytochrome-c reductase (NADH ferricyto-chrome B5 oxidoreductase (EC 1.6.2.2) (Omura & Takesue,1979); acid phosphatase (EC 3.1.3.2), using p-nitrophenylphosphate as substrate (Magun et al. 1982);'/J-jV-acetyl-glucosaminidase (EC 3.2.1.20) by a modification of themethod reported by Sellinger et al. (1960). Assay tubescontained 0 1 M-sodium acetate buffer, pH 5-0, 0-1 % TritonX-100, 2-2 j«nol/>-nitrophenyl /S-Ar-acetylglucosaminide, and100[i\ of sample. They were incubated at 37°C for 60min,the reaction stopped and colour produced by the addition oflml glycine buffer (133 mM-glycine, 83 mM-sodium carbon-ate, 67mM-NaCl, pH 10-7) and the absorbance read at400 nm. Enzyme activities were measured in 100 /-tl of gradi-ent fractions and 25 ̂ 1 of nuclear pellet or lysis supernatantand expressed as % activity of total cellular homogenate.

Electron microscopyFilters from the bottom of gradients were cut into two halves,folded, clamped in 3 mm diameter tubing and processed forelectron microscopy. Filters were fixed in dilute Karnovskyfluid (Karnovsky, 1965), postfixed in 2% osmium tetroxide,dehydrated and embedded in Epon without the tubing.Sections were stained with aqueous uranyl acetate and leadcitrate (Reynolds, 1963) and examined in a Philips 301electron microscope at 80 kV.

Endosome isolation in A431 cells 497

Alternatively, material was scraped from the filter in 52%sucrose solution, fixed in dilute Karnovsky fluid and appliedto poly-L-lysine-coated coverslips at room temperature over-night. The coverslips were air dried, washed in distilledwater and dried thoroughly before rotary shadowing withplatinum/carbon in a Balzers Electron Beam Evaporationequipment EVM (Balzers, FL-9496 Furstentum, Liechten-stein). An additional supporting layer of carbon was alsoapplied. The coated material was floated off the coverslipswith hydrofluoric acid, rinsed in distilled water and mounteddirectly onto copper grids.

SDS-polyacrylamide gel analysis of gradientfractionsTen confluent dishes of A431 cells were incubated for 18 hwith a total of 150^Ci L-[3sS]methionine in methionine-freemedium. The endosome fraction from the sucrose gradientsand a sample of PNS were precipitated in ice-cold 20%trichloroacetic acid (TCA) for 30 min. Pellets were formed bya 30-8 centrifugation at 8000 g. The pellets were resuspendedand incubated in acetone at — 20°C for 1 h. The precipitateswere pelleted and finally resuspended in sample buffer.

For comparison with the plasma membrane, a PNS wasprepared from a dish of A431 cells that had been surfaceiodinated using lactoperoxidase and glucose oxidase by themethod described by Hubbard & Cohn (1975). Samples wereTCA/acetone precipitated as described above.

Immunoprecipitates were prepared by diluting up to 500 /ilof PNS or endosome fraction with 1 ml of 200 mM-Tris • HC1,150mM-NaCl, 1 mM-EDTA (NET buffer) containing 1 mM-PMSF and 1 % NP40 and incubating with a 1:1000 dilutionof B3/25 antibody overnight at 4°C. Normal rabbit serumwas used as a control; 25—50 t̂l packed protein A-SepharoseCL-4B beads (Pharmacia Fine Chemicals, Uppsala 1, Swe-den) were added for 4 h at 4CC with gentle mixing. The beadswere removed by centrifugation at 300 g for 5 min and washedthree times in NET buffer+NP40 before finally resuspend-ing in sample buffer.

SDS-polyacrylamide gel electrophoresis (PAGE) usingreducing conditions was performed according to Maizel(1971). Gels were subsequently incubated in Amplify (Amer-sham International pic, UK) for 1 h, dried, and exposed toFuji RX film (Fuji Photo Film Co., Japan) at -70°C backedby an intensifying screen.

Results

Density shift of an endocytic compartment usingcolloidal gold

A431 cells were incubated with either 125I-labelledgold-B3/25 or 12sI-labelled B3/25 for 1 h at 20°C andlysed as described in Materials and methods. Thepostnuclear supernatant contained 70-7% ± 6-2 (stan-dard deviation; n = 10) of the radioactivity (theremainder was not released from the nuclear pellet)and was fractionated on sucrose gradients. Fraction 1(density-shifted filter fraction), at a density> l - 2 3 g m r l contained 27% of the 12iI-labelledgold-B3/25 originally associated with the cells (i.e. %

homogenate) compared with only 0-2% of 1Z5I-labelledB3/25 without the gold complex. The majority of 125I-labelled B3/25 loaded onto the gradient was recoveredin a sharp peak in fraction 11 (density 1-128 ml"1) (Fig.1A). The colloidal gold produced a significant densityshift of at least 0-102gml"1. Free 125I added to thegradient peaked sharply in fraction 13 (profile notshown). It has been shown previously that when A431cells are incubated with gold-B3/25 for 1 h at 20°C,most of the gold complex remains associated with thetransferrin receptor (Hopkins & Trowbridge, 1983)and therefore, we would expect the majority of 125I-labelled gold-B3/25 used in these experiments to alsoremain membrane-associated.

The effect of trypsin treatment on l25I-labelled gold—B3/25 distribution

When cell fractionation was performed without theaddition of trypsin to the homogenate, the yield of I2SI-labelled gold-B3/25 at the bottom of the gradient wassubstantially less; approximately 5-5% (Fig- 1A).However, these conditions allowed only 49% ± 7-4 ofthe cell-associated radioactivity to be recovered in thePNS. This could be increased to 73% ±13-4 bywarming the cell homogenate to 37°C for 3 min.Warming increased the yield of radioactivity in fraction1 to 17-9% but also gave peaks of radioactivitythroughout the gradient. Trypsin treatment thereforehad two benefits: (1) it increased the release of thegold-labelled compartment to the PNS; and (2) itincreased the proportion of the compartment in thePNS that shifts to fraction 1.

The appearance of fraction 1 prepared with andwithout trypsin treatment was studied by electronmicroscopy of carbon-shadowed samples on coverslips.The untrypsinized fraction consisted of large, gold-containing globular elements entangled in filamentousmaterial (Fig. 2). Trypsin treatment removed thefilamentous material completely, leaving areas of gold-containing tubular and vacuolar elements, which oc-casionally appeared to be connected. Thereafter, tryp-sin was used routinely in the preparation of postnuclearsupernatants.

The effectiveness of the trypsin treatment in thepreparation of a PNS may be an important factor indetermining which part of the endosome system we areable to isolate, as well as increasing the yield. Webelieve trypsin treatment to be useful in degrading thecytoplasmic filaments in these cells, thus permittingmore efficient dispersal and fractionation of subcellularorganelles. This may be particularly useful in isolatingparts of the juxtanuclear endosome compartment, asour own recent morphological studies show this to beenmeshed in a pericentriolar mass of filaments.

498 J. Beardmore et al.

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Fig. 1. A. The recovery of 12SI-labelled B3/25 and1Z5I-labelled gold-B3/25 on sucrose gradients. Lysissupernatants were prepared from A431 cells incubated for1 h at 20°C with either 125I-labelled B3/25 or 125I-labelledgold-B3/25. Gradient load = lml. ( • • ) lzsI-labelledgold-B3/25, trypsin-treated lysate (seven experiments) and(O O) no trypsin treatment (four experiments);(A A) 1Z5I-labelled B3/25, trypsin-treated lysate (threeexperiments). B. The distribution of [125I]transferrin and12iI-labelled B3/25 on sucrose gradients. Cells wereincubated for 1 h at 20°C in the presence of lOOngml"1

[12sI]transferrin or 12sI-labelled B3/25 and gold-B3/25 andchased for 30min at 20 °C withGradient load = 1 ml. (<(O----O) [1Z5I]transferrin.

transferrin.125"l-labelledB3/25;

The effects of a range of trypsin concentrations(0-100 fig ml"1) on samples of PNS from L-[35S]meth-ionine-labelled cells were studied using SDS-PAGE

2AFig. 2. Whole-mount preparation of endosome-enrichedfraction from non-trypsinized cell homogenate. A. Sampleswere rotary shadowed with carbon/platinum. X 15 000.Note that the membrane elements are entangled in afilamentous network. Arrows: gold containing elements,but the gold is not clearly visible at this magnification.B. Whole-mount of isolated gold-containing endosomeelement from trypsin-treated homogenate enlarged todisplay gold label. X60000. Bar, 0-2 jtm.

electrophoresis (results not shown). The resultsshowed that at a concentration of 0-5j(gml~', therewas a slight lessening of intensity of two bands around220-250 (xlO3)yV/r. At concentrations of 10 jig ml"1

and above several proteins over a wide range ofmolecular weights were seen to disappear progress-ively. We conclude therefore that at the concentrationof trypsin routinely used (0-5 jig ml"1) the proteolysisof most cellular proteins is negligible.

Co-localization of [l25I]tra7isferrin, l25I-labelledB3/2S and gold-B3/25 in endosome-enrichedfractions

Gradient analysis of cell supernatants from cells incu-bated with 125I-labelled B3/25 demonstrated the pos-ition of an endosome compartment from A431 cells thathad not been density-modified (Fig. 1A). By incu-bating cells simultaneously with [125I]transferrin or125I-labelled B3/25 and gold-B3/25 for 1 h at 20°C,followed by a 30-min chase with 10 jig ml"1 transferrinat 20°C, we were able to show that [125I]transferrin(12% homogenate) and 12iI-labelled B3/25 (11%homogenate; presumably still bound to the transferrinreceptor) is also routed into the density-modifiedendosome compartment highly enriched in fraction 1(F.g. IB).

Endosome isolation in A431 cells 499

Characterization of gradient fractionsThe distribution of various marker enzymes withrespect to 125I-labelled gold-B3/25 on sucrose gradi-ents is shown in Fig. 3. We obtained good separation ofmost of the components on the gradient and they weredistributed in the density ranges expected comparedwith those reported in the literature (Beaufay et al.1974). All marker enzymes studied were present only invery low or undetectable amounts (0-09% of homogen-ate) in fraction 1, where the peak of 125I-labelledgold-B3/25 occurred. Enzyme activities were found tobe equivalent in non-trypsinized and trypsinizedsamples and so we believe our values to be a truerepresentation of the lack of contamination in fraction1.

To obtain an independent, non-enzymic plasmamembrane marker, [125I]EGF was allowed to bind tothe surface of cells for 30 min at 4°C, after the cells hadbeen exposed to gold-B3/25 for l h at 20°C. Theposition of the surface-bound [12SI]EGF on the sucrosegradients closely parallels that for 5' nucleotidase (Fig.3), suggesting that these two markers are following thedistribution of plasma membrane. Both markers peakin fractions 9 and 10, with only very low levelsdetectable in the denser gradient fractions and less than0-2 % detectable in fraction 1. Between 85 and 100 % ofthe [12SI]EGF in the gradient fractions could beprecipitated with ice-cold 10 % trichloroaceticacid/l % phosphotungstic acid, demonstrating that thelabelled peptide remains intact during the lysis pro-cedure. When [12SI]EGF was added to gold-B3/25-containing lysis supernatants and then fractionated onsucrose gradients, approximately 0-2% of the totalradioactivity was present in fraction 1, with the ma-jority of the radioactivity loaded being recovered as asharp peak in fractions 12 and 13, i.e. in the loadvolume (results not shown).

The yield and relative specific activities (RSA) of thevarious enzyme and radioactive markers in gradientfraction 1 are shown in Table 1. There is a significant

Fig. 3. Distribution of marker enzymes and radioactivemarkers in lysis supernatants. Cells, labelled withL-[35S]methionine or [methyl-2H]cho\'\ne chlorideovernight, were incubated with gold-B3/25 for 1 h at20 °C, lysed, and fractionated on continuous sucrosegradients. Gradient load = 1 ml. The distribution of markerenzymes was determined on parallel gradients using lysatesprepared from 125I-labelled gold-B3/25 labelled cells.Some cells incubated for 1 h at 20°C with gold-B3/25 werefurther incubated at 4°C with 10 ng [125I]EGF/dish for30 min, to act as a cell surface marker during fractionation.Values are the mean of three experiments. Recoveries were70-87% for 125I-labelled gold-B3/25, 80-100% for markerenzymes and 70—100% for L-[35S]methionine and [methyl-3H]choline chloride.

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500 J. Beardmore et al.

Table 1. Composition of gradient fraction 1(endosome enriched)

Marker

L-[35S]methionine125I-labelled gold-B3/255' Nucleotidase[125I]EGFGalactosyl-transferaseNADH-cytochrome-c reductase/J-A'-acetyl-glucosamimdaseAcid phosphataseSuccinate-INT reductase[Afef/iy/-3H]choline chloride

% Homogenate

0-28 ±0-04626-8 ±6-10-15 ±0-0015 ±0-010-25 ±0-20-0

0-47 ± 0-030-9 ±0-380'3±0-42

0-15 ±0-05

RSA

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% Homogenate values are means of all experiments ±S.D.L-[35S]methionine incorporation was used to assess the proteincontent. Relative specific activity (RSA) represents the ratio9between the percentage of marker and that of protein ( L - [ 3 5 S ] -methionine) with respect to the homogenate, as defined byfeeaufay & Amar-Costecec (1976).

difference in the yield of protein (0-28 %) and phos-pholipid (0-15%) in this fraction. Apart from the 88-fold enrichment of I-labelled gold—B3/Z5 over thehomogenate, only the lysosomal markers show a slightenrichment in this fraction. In all recent experimentswe have routinely obtained an enrichment of 12SI-labelled gold—B3/25 to protein marker in fraction 1 of200-fold ±17 (n = 10).

Morphology of fraction 1

Previous papers on studies of the pathway of processingof gold-B3/25 by A431 cells (Hopkins & Trowbridge,1983; Hopkins, 19836) have described an endosomalsystem comprising tubular cisternae, vacuolar elementsand multivesicular bodies, the latter being predomi-nant in the juxtanuclear area of the cell, where they aresurrounded by cisternae. All of the components de-scribed became labelled with gold-B3/25 when incu-bated at 20 °C, the cisternal elements often being themost heavily labelled.

Filters from the bottom of the sucrose gradients usedto isolate an endosome fraction, identical to thoseanalysed in Fig. 3, were processed for electron mi-croscopy. Thin sections across the filters showed thepresence of a highly purified population of small, gold-containing elements (Fig. 4). These elements vary insize and shape, appearing as small vesicles containingonly a few gold particles, and tubular elements or largervesicles, containing many gold particles. Counting theprofiles showed 80 % (n = 600) or more of the mem-brane-bound elements contain gold. This value is likelyto be an underestimate because some apparently emptymembrane profiles may contain gold particles in othersection planes. Rough estimates show that 25 % or lessof the total number of gold particles seen in thin sectionare not enclosed within membrane profiles and may be

free gold. Gold within membrane elements is oftenseen in close apposition to membrane, especially in thelarger vesicles, suggesting that it is still bound to thetransferrin receptor via the B3/25 antibody. Theminor contaminants of this preparation could be ident-ified predominantly as microfilaments. Pieces of gold-labelled smooth membrane and the occasional lyso-some-like structure were also observed.

Comparison of endosome-enriched and plasmamembrane-rich fractions on SDS-polyacrylamide gelsThe endosome-enriched fraction and PNS preparedfrom L-[3sS]methionine-labelled cells and l2sI-labelledplasma membrane containing PNS were analysed bySDS-PAGE and fluorography (Fig. 5). The L-[3SS]methionine-labelled endosome fraction can beseen to contain at least 10 major polypeptide bands andseveral minor bands ranging in molecular weight fromapproximately 200 to 14 (X103) A/r. This fraction has asimpler polypeptide profile than the L-[3SS]meth-ionine-labelled PNS and 12sI-labelled plasma mem-brane fractions. Three polypeptides (approx. 200, 63,15 (XlO3)Mr) appear to be enriched in the endosomefraction when compared with the PNS. One of these,the 200xl03A/r protein, appears to be stronglyrepresented in the 12sI-labelled plasma membranepreparation.

The transferrin receptor was immunoprecipitatedfrom both the L-[ SJmethionine-labelled endosomefraction and PNS using the B3/25 antibody (Fig. 6)and demonstrates a band at an Mr of around 90X 103,which is the reduced subunit of the disulphide-bondedreceptor dimer (Trowbridge & Omary, 1981). A minorcontaminant of the immunoprecipitates was the200xl03Afr protein, which is strongly represented ingels of the PNS and endosome fraction.

Discussion

In this study we have used colloidal gold complexeswith an antibody to the human transferrin receptor toisolate elements from the endosome pathway in A431cells. We have been able to demonstrate a concentrationof transferrin receptors in the endosome-enriched frac-tion by using a pulse-chase protocol to introduce 12SI-labelled B3/25 into gold-loaded elements at 20°C. Webelieve that the presence of a high proportion of the1Z5I-labelled B3/25 in the gold-containing fraction canonly be explained by its affinity for the transferrinreceptor, and it seems likely that the receptor is aconsistent constituent of the isolated endosome frac-tions. This is confirmed by our ability to immuno-precipitate the receptor from this fraction.

The internalization of large amounts of gold-B3/25by A431 cells has enabled us to obtain a high yield of ahighly purified endosome fraction by using a single

Endosome isolation in A431 cells 501

•4

* -

> 4

4A B. v . •».

Fig. 4. Appearance of the enriched endosome fraction centrifuged onto supporting filter. A,B and D—I. 60nm thicksections; C, an 800nm thick section. Bar, 0'2/jm. A-C. Typical low magnification (X20000) cross-sectional views of thefolded filter surface displaying loosely packed endosomal elements. In addition to the small vesicular structures there aremore elaborate tubular elements and larger vesicles (small arrows). The majority of the membrane-bound elements containgold. C. Thick sections are better able to show the extent of the gold labelling in the denser organelles (large arrows).D—I. Higher magnification (D-G approx. X 100000; H,I approx. X70 000 and 83 000, respectively) micrographs of thefilter surface showing representative endosomal elements in the fraction.

502 J. Beardmore et al.

fractionation step on sucrose gradients. In particular,the density modification procedure allowed us to separ-ate the endosome efficiently from the plasma mem-brane, which would normally have a similar density.

1 2 3

/WrX1(T3

2000'

1000'

690-

46 0

30-OH

14 3'

Fig. 5. Fluorograph of L-[35S]methionine-labelledendosome-enriched fraction, PNS and I-labelled plasmamembrane preparation from A431 cells. A S % to 12%gradient gel was used under reducing conditions. Lane 1 isL-[35S]methionine-labelled endosome-enriched fraction(sample load = total endosome-enriched fraction (fraction1) from approx. 60X106 cells); lane 2 is L-[3SS]methionine-labelled PNS ( lOxlO 6 cells); lane 3 is 125I-labelled plasmamembrane (3X106 cells).

}~3

200

100

69

46-

3O

Fig. 6. Fluorograph of the immunoprecipitated transfemnreceptor from the endosome fraction and PNS preparedfrom L-[35S]methionine-labelled cells. Lane 1, PNS(3-5X106 cells), B3/25 immunoprecipitation; lane 2,endosome fraction (60X106 cells), B3/25immunoprecipitation.

The density of the unmodified endosome compart-ment in A431 cells as marked by l25I-labelled B3/25content was 1-12—l-13gml~', which is in agreementwith that for endosome-enriched fractions preparedwithout a density modifying step from other cellsystems (Dicksone* al. 1983; Quintart e/ al. 1984; Wall& Hubbard, 1985). In these systems the enrichment ofendosome marker is relatively low (20- to 63-fold). Byshifting the density of endosomes to >l -24gml~ ' wewere able to increase the enrichment of endosomemarker in the isolated endosome fraction to 200-fold,which is in good agreement with the degree of puri-fication obtained by density-modifying endosomeelements in rat liver (Quintart et al. 1984).

Characterization of the endosome-enriched fractionfrom A431 cells demonstrated that it contains 27% ofthe cell-associated 125I-labelled gold-B3/25 (the ma-jority of which is membrane-associated) and only verylow levels of the marker enzymes for other subcellularorganelles (0-0-9%). The levels of marker enzymesreported from endosome-enriched fractions preparedfrom other cell types varied considerably (Lamb et al.1983; Luzio & Stanley, 1983; Quintart et al. 1984;Saemark et al. 1985; Wall & Hubbard, 1985). How-ever, in endosome fractions prepared from rat liver(Quintart et al. 1984; Wall & Hubbard, 1985) and KBcells (Dickson et al. 1983) the co-localization of markerenzymes with endosome marker has also been reportedto be very low.

The morphology of fraction 1 showed that it maycontain low levels of plasma membrane contamination.Because a pulse-chase protocol was not used in theseexperiments, some gold-B3/25 will have been boundat the cell surface when the cells were lysed and thiscould explain the presence of some sheets of gold-labelled membrane in the endosome-enriched frac-tions. However, our attempts to label only the cellsurface by incubating with gold-B3/25 at 4°C were noteffective in shifting the density of plasma membrane. Ifplasma membrane is a contaminant, the very low levelsof EGF receptor and 5' nucleotidase detected in thisfraction suggest that the transferrin receptor-contain-ing regions have been preferentially removed from thecell surface.

Electron microscopy of the endosome-enriched frac-tion showed a predominance of gold-labelled tubulesand small vesicles, and a smaller number of multives-icular bodies, all similar to the elements seen in theintact cell. It is possible that the number of multives-icular bodies is lower than expected because they donot contain enough gold-B3/25 to shift their density tofraction 1. We may, therefore, be isolating a specificpart of the endocytic pathway by the experimentalprocedure used. The elements isolated by loading withgold—B3/25 are also dependent upon the distribution

Endosome isolation in A431 cells 503

of the transferrin receptors within the endocytic path-way, and since there is evidence from earlier studiesthat these receptors are preferentially distributed in thecisternal elements in these cells (Hopkins, 19836) thismay explain the predominance of tubular elementswithin the isolated fraction.

We used L-[35S]methionine-labelled cells to producean endosome-enriched fraction so that its polypeptidecomposition could be studied on SDS-polyacrylamidegels. The use of L-[35S]methionine ensures that we arecomparing only newly synthesized cellular proteins,but this method excludes proteins that either do notcontain methionine or have a very slow turnover rate.However, comparison with similar fractions on gelsstained with Coomassie Blue suggests that this is not asignificant problem, at least for the major polypeptidebands (unpublished observations). Both the domi-nance of high molecular weight bands on the reducinggel and the results from a titration experiment withdifferent concentrations of trypsin suggest that thetrypsin treatment used in the lysis procedure does notcause extensive proteolysis of the protein constituentsof the endosome membrane. We confirm the obser-vation that endosome membrane is simpler in polypep-tide composition than plasma membrane, previouslyreported by Watts (1984) using HepG2 cells andDickson et al. (1983) using KB cells. Different label-ling methods were used to identify the protein bands inthe endosome and plasma membrane fractions and so adirect comparison between the two fractions may notbe valid. However, the L-[35S]methionine-labelledpost-nuclear supernatant from A431 cells showed thatthere were protein bands at the same molecular weightsas those seen in both the L-[3SS]methionine-labelledendosome and the 12sI-labelled plasma membranepreparations, suggesting that a comparison betweenthe two differently labelled samples is meaningful. Weare also able to show that the endosome fraction has adifferent polypeptide composition from that of the 125I-labelled plasma membrane preparation, although sev-eral polypeptides are common to both. The identity ofthe proteins present in, or associated with, endosomemembranes is not known. We can, however, identifythe transferrin receptor by immunoprecipitation. Webelieve that the 43-46 (X 103)Mr polypeptide presentin both endosome-enriched and iodinated plasmamembrane fractions is actin, because microfilamentsare seen as a minor contaminant of our isolatedfraction. A 43xlO3Mr protein was also found in anendosome-enriched preparation from KB cells (Dick-son et al. 1984).

There are several proteins at around 35xlO3Afr inthe endosome-enriched fraction from A431 cells. Saw-yer & Cohen (1985) have reported that a 35xlO3Afr

protein is a calcium-dependent substrate for the EGFreceptor/kinase in intact A431 cells and it is of interest

that enhanced phosphorylation of this protein can bedetected only in cells exposed to EGF for periods ofmore than 30 min (a time when receptor-ligand intern-alization into the juxtanuclear endosome becomes sig-nificant). It will be interesting to see if the 35 X 103Mr

proteins of the isolated endosome are substrates for theEGF receptor kinase.

By incubating cells with both [1Z5I]transferrin andgold-B3/25 we produced an endosome fraction thatcontained 12% of the cell-associated [125I]transferrinand 27% of the gold-B3/25, suggesting that we areisolating a functional compartment through which bothtransferrin and its receptor are routed. When preparingbulk endosome fractions we have observed that the tophalf of the sucrose gradient can appear pink, confirm-ing our belief that we label only a subfraction of theendosome system sufficiently heavily to cause a signifi-cant shift in its density. This is likely to be the mainreason why only 12% of the [lzsI]transferrin is in thehighly purified endosome fraction at the bottom of thegradient.

The experimental conditions used to load the A431cells with gold-B3/25 in this paper (continuous ex-posure for 1 h at 20 °C) would label the endosomeelements throughout the cell, but we would expectfrom our morphological analysis of whole cells that thejuxtanuclear compartment would be the most heavilylabelled under these conditions. We feel, therefore,that our endosome-enriched fraction consists predomi-nantly of juxtanuclear endosome elements throughwhich the transferrin receptor is being processed. Byloading cells for different times with gold—anti-trans-ferrin receptor antibody complexes we should now beable to isolate, preferentially, other parts of the endo-some pathway.

The role of the prelysosomal endosome compart-ment in processing internalized cell surface receptorsand their ligands is now well established (Miller et al.1986; Van Renswoude et al. 1982; Willingham et al.1983; Yamishiro et al. 1984). However, the moleculardetails of the mechanisms responsible for this process-ing are unclear. These mechanisms include the selec-tive routing of internalized receptor proteins, thetranslocation of internalized ligands across the endo-some membrane to the cytoplasm, and the transductionof mitogenic and other growth-related signals. Theavailability of the endosome preparation we havedeveloped should permit all of these questions to beaddressed directly.

Throughout this work Adele Gibson, Adrian Walsh andCarole Thomas provided excellent technical assistance,which was greatly appreciated. The authors thank RobertoSolari for his help and advice.

Julia Beardmore was supported by a short-term EMBOFellowship and a grant from the Medical Research Council.

504 J. Beardmore et al.

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(Received 22 December 1986 - Accepted 12 February 1987)

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