Vol. 5, 41 1-417, April 1994 Cell Growth & Differentiation 411

The Protein Product of the Oncogene bcl-2 Is aComponent of the Nuclear Envelope, theEndoplasmic Reticulum, and the Outer

. . 1Mitochondrial Membrane

Trevor Lithgow,2 Rosemary van Driel, John F. Bertram,and Andreas Strasser3

Department of Biochemistry, La Trobe University, Bundoora 3083 IT. LI;The Walter and Eliza Hall Institute of Medical Research, Post Office,

Royal Melbourne Hospital, victoria 3050 IR. v. D., A. 5.1; andDepartment of Anatomy and Cell Biology, University of Melbourne,Parkville, Victoria 3052 Ii. F. B.l, Australia

Abstrad

The protein produd of the oncogene bcl-2 is a potentinhibitor of apoptotic cell death. The Bcl-2 protein hasvariously been reported to reside in the nuclearenvelope and endoplasmic reticulum or exclusively inthe inner membrane of mitochondria. We thereforeundertook a detailed analysis of the intracellulardistribution of Bcl-2 by immunofluorescence,immunogold eledron microscopy, and subcellularfradionation in three mouse cell lines expressing ahuman bcl-2 transgene and measured its importationinto isolated mitochondria. By these methods, theprotein was localized to the nuclear envelope, theendoplasmic reticulum, and the outer mitochondrialmembrane. Any proposal for the mechanism by whichBcI-2 inhibits apoptosis must therefore accommodate thefad that Bcl-2 localizes to cytoplasmic membranesfacing the cytosol.

Introdudion

Apoptotic death is a physiological process responsible forthe removal ofobsolete as well as potentially dangerous cellsof multiple lineages (1-4). Various hemopoietic and neuro-nal cells undergo apoptosis in response to deprivation ofspecific cytokines (3, 5), and lymphoid cells can be inducedto die by a variety of physiological as well as experimentallyapplied stresses (6, 7). Constitutive, high level expression ofbc!-2 enhances the survival of hemopoietic and neuronalcells cultured in the absence of growth factors (8-14) andinhibits apoptosis of B- and T-Iymphocytes under variousconditions of stress in vitro as well as in vivo (1 5-23). Thebiochemical mechanism by which bc!-2 expression pne-

Received 11/15/93; revised 1/9/94; accepted 1/27/94.

I This work was supported by the National Health and Medical ResearchCouncil of Australia and by U.S. National Cancer Institute Grant CA43540 toDr. S. Cory. T. L. was supported by a grant from the Australian Research

Council to Dr. N. J. Hoogenraad, and A. S. was supported by fellowships fromthe Leukemia Society ofAmerica and the Swiss National Science Foundation.2 Present address: Department of Biochemistry, Biozentrum, Basel CH-4056,

Switzerland.3 To whom requests for reprints should be addressed, at the Walter and Eliza

Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital,Victoria 3050, Australia.

vents apoptosis is presently unknown, and the location of the26 kDa4 Bcl-2 protein within the cell has been controversial.On the basis of subcellular fractionation and immunofluo-rescence staining, one group has reported that it is associated

exclusively with the mitochondnial inner membrane (10).However, other experiments using similar experimentalmethods suggested that the protein is associated with theendoplasmic reticulum and the nuclear membrane and isexposed to the cytosol (24-26). In addition, three recentstudies by immunofluonescence and/or electron microscopyfound Bcl-2 in the nuclear membrane and the endoplasmicreticulum as well as in mitochondnia (27-29). Because thesubcellulan location of Bcl-2 must relate to its function, it isimportant to resolve whether on not Bcl-2 is enclosed withinmitochondnia.

To address this question, we have undertaken an analysisusing subcellulan fractionation, immunofluonescence mi-croscopy, and immunogold electron microscopy of threeindependent mouse cell lines infected with a retrovirus cx-pressing a human bc!-2 cDNA. Our results establish that theintracellular location of Bcl-2 is not cell type specific; BcI-2is a component of the nuclear envelope, the endoplasmicreticulum, and the mitochondnial outer membrane in allthree cell types. In addition, using a well characterized assaysystem for mitochondnial protein importation (30, 31), wedemonstrate that the protein can be inserted into the outenmembrane of isolated m itochondnia.

Results

Immunofluorescence. Four different mouse cell lines,namely FDC-P1 myeloid cells, WEHI-23i B-Iymphomacells, B6.2.i 6.BW2 T-hybnidoma cells (hereafter referred toas B6.BW2 T-cells), and L929 fibroblasts were infected witha netnovinus expressing a human bc!-2 cDNA and the neorgene (hereafter referred to as bcl-2/neo virus) or, as a control,with a netrovinus expressing only the neor gene (hereafterreferred to as neo virus). Immunofluonescence staining wasperformed on multiple independent clones to study the sub-cellular localization ofthe BcI-2 protein. The staining pattern

obtained with a monoclonal antibody specific fonthe humanBcl-2 protein cleanly differed from that obtained with an an-tisenum containing autoantibodies specific for the intrami-tochondnial enzyme pynuvate dehydnogenase complex E2(Fig. 1). The former labeled only discrete, punctate structuresin the cytoplasm. In contnast, Bcl-2 staining was widespreadthroughout the cytoplasm in all three cell types (Fig. 1). Asimilar staining pattern was also seen in WEHI-23i mouse

4 The abbreviations used are: kDa, kilodalton(s); cDNA, complementaryDNA; B6.BW2 T-cells, B6.2.16.BW2 T-hybridoma cells; nec’, neomycin re-sistance; preOTC, precursor ofornithine transcarbamylase; SDS, sodium do-

decyl sulfate; IL-3, interleukin 3.

Fig. 1. Immunofluorescence analysis of Bcl-2 protein localization. L929 fi-

broblasts, B6.2.16.BW2 T-hybridoma cells (B6.BW), and FDC-P1 myeloidcells infected with a bc!-2/neo virus were stained either with the monoclonalantibody BcI-2-100, which is specific for the human Bcl-2 protein (45), orwith serum from a primary biliary cirrhosis patient with a high titer of au-toantibodies directed against the mitochondrial pyruvate dehydrogenase E2

complex (46). These micrographs are representative of 5-10 independentclones of each cell line.

412 Intracellular Distribution of Bcl-2

Pyruvafe

B-Iymphoma cells infected with the bc!-2/neo virus; in thehuman diffuse histiocytic B-Iymphoma cell line SU-DHL-4,which overexpresses BcI-2 as a result of its t(i 4;i 8) chro-mosome translocation (24); and in nesting or activated B- andT-lymphocytes from Ep-bcl-2 transgenic mice (20). Bcl-2 isknown to inhibit apoptosis in cytokmne-depnived FDC-P1

cells (8) and in glucocorticoid-treated B6.BW2 T-cells.5 Fur-thenmore, a constitutive high level of Bcl-2 protects FDC-P1,

B6.BW2, WEHI-231, and L929 cells from hyperthenmia-induced apoptosis.6 Importantly, no major change in theBcl-2 staining pattern was observed in these circumstances.These data suggest that the Bcl-2 protein is not restricted tomitochondnia but might also be present in other locationswithin the cytoplasm.

Subcellular Fractionation. In an attempt to obtain moredetailed information on Bcl-2 protein localization, weturned to subcellulan fractionation. The internal membranesfrom each ofthe bcl-2/neo virus-infected cell lines were pu-

S T. Lithgow, R. van Driel, J. F. Bertram, and A. Strasser, unpublished ob-

servations.6 A. Strasser and R. L. Anderson, submitted for publication.

nified by sucrose gradient centnifugation, and, in the case of

the FDC-Pi cells, mitochondnia were further subfractionatedinto outer and inner membranes. Some ofthe ruptured outer

membrane can be purified from homogenized mitochondnia

as membrane vesicles; the remainder is associated with theintact inner mitochondnial membrane in the “mitoplast”fraction (see Ref. 32). The presence of Bcl-2 in the various

membrane fractions was assessed by immunobbotting, and

Phosphorlmager analysis was used to quantitate the level ofBcl-2 as a proportion of the protein content. The relativepurity of the fractions was assessed by measurement of spe-

cific activities of two marker enzymes, namely the inner mi-

tochondnial membrane marker cytochnome c oxidase andthe outer mitochondnial membrane marker monoamine oxi-

dase. Human Bcl-2 protein was strongly expressed in allthree lines infected with the bd-2/neo virus but not in con-trol neo virus-infected cells (Fig. 2, A and B, Lanes 1 and 2).In concordance with the work of Hockenbery et a!. (10),Bcl-2 protein was clearly detected in the mitochondnial frac-tion. However, Bcb-2 was not restricted to mitochondnia butwas also present in the microsomal fraction and in thenuclear envelope (Fig. 2, A and B). Although Bcl-2 was alsopresent in the pore complex fraction of bc!-2/neo virus-infected FDC-P1 myeboid cells (Fig. 2A), no enrichment of

the protein was evident in this subfraction of the nuclearenvelope. The presence of Bcl-2 in the nuclear envelope andmicrosomal fractions cannot be accounted for by mitochon-

dnial contamination, since no monoamine oxidase or cyto-chrome c oxidase activity could be detected in these frac-tions (Fig. 2A). Hence, Bcl-2 protein appears to be a bonafide component of the nuclear envelope and the endoplas-

mic reticulum.Bcl-2 protein was detected both in the outer mitochon-

dnial membrane vesicles and in mitoplasts (Fig. 2A). Signifi-cantly, since the former was devoid of cytochnome c oxidaseactivity, but enriched in both monoamine oxidase activity

and Bcl-2, the presence of Bcl-2 in the outer membranevesicles cannot be accounted for by contamination with in-ncr mitochondnial membranes. However, the amount ofBcl-2 recovered with mitoplasts correlated with the level ofmonoamine oxidase, consistent with substantial contami-nation of this fraction by outer mitochondnial membranes.These data suggested that mitochondnial Bcl-2 is confined to

the outer membrane.Electron Microscopy. To further define the subcellular bo-

calization of BcI-2, we used immunogold electron micros-copy. Immunogold labeling was significantly greater (3-10-fold, P-value at least 0.001 ) in FDC-Pi cells infected with the

bc!-2/neo virus than in the control cells (Table 1 ). Most of thegold particles in the former were associated with cytoplas-mic membranes, whereas the background signal in the con-

trol cells was more randomly distributed (Table 1 ). Specificlabeling was detected on mitochondnia, the nuclear enve-lope (Fig. 3, A and B), and the endoplasmic reticulum (Fig.3C). On all ofthese organdIes, Bcl-2 protein appeared to belocalized to the cytosolic surface. Significantly, we detectedno labeling associated with the mitochondnial inner mem-

brane (Fig. 3A). Quantitative analysis of the subcellular dis-tnibution of the gold particles indicated that 1 7% were as-sociated with the outer mitochondniab membrane, 34% withthe nuclear envelope, and 49% with other cytoplasmic

membranes including the endoplasmic reticulum (Table 2).A similar distribution of the Bcl-2 protein was found in dcc-

tron micrographs prepared from immunogold-labeled

B6.2.16.BW2bC/-2/neo

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Cell Growth & Differentiation 413

A

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monocim,neox�dase I

NoI x � 0 f o � o � 0 J1.2 � 8 9 1.1 Inmo(/min/mg protein)

-� cytochrome c oxidase I

[�1N0I X � 0 � 0 j 0 � 0 I’�I � ‘� (nmot/m,nAngprotein)]

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Fig. 2. Localization of Bcl-2 protein by subcellular fractionation. FDC-P1 myeloid cells (A) or B6.2.16.BW2 T-hybridoma cells and L929 fibrohlasts infectedwith bcl-2/neo or neo virus (B) were analyzed for BcI-2 protein content by Western blotting using the monoclonal antibody Bcl-2-100. Lysates were prepared

and fractionated as described in “Materials and Methods.” Equal volumes of each fraction were loaded. The total protein concentration in each subcellular fractionwas measured, and the relative concentration of Bcl-2 protein was determined by Phosphorlmager analysis of 1251-Protein A bound to the blot membrane. The

enzymatic activity of monoamine oxidase and cytochrome c oxidase provided an indication of the relative purity of each subcellular fraction. For each cell line,no activity (<0.1 nmol/min/mg) of either enzyme could be detected in the cytosolic, nuclear envelope, or microsomal fraction. These results are typical of those

obtained in 2-4 independent experiments with each cell line. Molecular weights are indicated in kDa at left. ND, not determined; X, empty lane on the gel.

Table 1 Immunogold labeling density and percentage membrane distribution of Bcl-2 in FDC-P1 cells infected with bcl-2/neo or neo virus

FDC-P1 neo cells are FDC-P1 cells infected with the control neo virus. FDC-P1 bcl-2/neo cells are cloned FDC-P1 cells infectedtable summarizes the analysis of more than 100 micrographs for both cell lines (see “Materials and Methods”).

with teh bcl-2/neo virus. This

FDC-P1 bcl-2/neo

Organelle Labeling Membrane. . . . I

density” distribution / ‘

FDC-P1 neo

Labeling#{149}

density. .

Membrane distrtbut�on /,,

Nucleus 0.01 ‘ 72

Mitochondria 0.02 82

Other cytoplasmic membranes (including endoplasmic reticulum) 0.008 75

0.003

0.002

0.001

9

54

25

,, Labeling density is the number of immunogold particles/0.01 pm2 of sectioned organelle.I, Membrane distribution is percentage of total label on this organelle which was associated with membranes.‘ P-values are <0.001 for each organelle.

B6.BW2 T-cells and L929 fibroblasts infected with the bC!-2/neo virus (data not shown). These data are concordantwith those described above from immunofluorescence andsubcellulan fractionation.

Translocation and Importation of BcI-2 into Isolated Mi-tochondria. Chen-Levy and Cleary (25) have previouslydemonstrated that Bcb-2 protein can be inserted into isolatedmicrosomes. We tested whether Bcl-2 could be inserted intoisolated mitochondnia in a well characterized in vitro assayfor the importation of mitochondnial proteins (31 ). The pre-cursor ofonnithine transcarbamylase (preOTC), a mitochon-dnial enzyme which associates with the inner mitochondnialmembrane after proteolytic processing in the matrix (33),was used as a positive control. Both preOTC and Bcl-2 weresynthesized and radioactively labeled with lt5Slmethioninein a rabbit reticubocyte lysate translation system and thenincubated with mitochondnia isolated from rat liver. Mito-chondnia were then subfractionated into outer membranesand mitoplasts, and imported proteins were detected by flu-orography of SDS-polyacrylamide gels (Fig. 4). As expected(31 ), mature ornithinetranscarboxylase was recovered in the

mitoplast fraction, even when the mitochondnia had beentreated with Proteinase K (Fig. 4, Lanes 4 and 6). In contrast,Bcl-2 protein was found both in the outer mitochondrialmembrane fraction as well as in mitoplasts (Fig. 4, Lanes 3and 4). The presence of BcI-2 in mitoplasts presumably ne-suited from the presence of contaminating outer mitochon-dniab membranes in this fraction. Consistent with this notion,BcI-2 protein was not detected in mitoplasts or in the mi-tochondnial outer membrane fraction purified from Protein-ase K-treated mitochondnia (Fig. 4, Lanes 5 and 6). We con-cludethat Bcl-2 can be imported in vitrospecifically into theouter membrane of isolated mitochondnia.

Discussion

We have used three separate techniques, namely immun-ofluorescence, immunogold electron microscopy, and sub-cellular fractionation combined with immunobbotting, to de-termine the subcellular localization of Bcl-2 protein inmouse myeboid, T-bymphoid, and fibroblast cell lines in-fected with a bc!-2/neo virus. The results established that

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414 Intracellular Distribution of Bcl-2

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Fig. 3. Detection of Bcl-2 protein by immunogold electron microscopy.Electron micrographs illustrating localization of Bcl-2 protein on (A) outer

membrane of mitochondria and on the nuclear envelope (X 91,000), (B)

nuclear envelope (X 107,800), and (C) other cytoplasmic membranes, pos-sibly endoplasmic reticulum (X 143,500) of FDC-P1 myeloid cells infectedwith bcl-2/neo virus. These micrographs are representative of more than 100

examined from 3 independent experiments.

Bcl-2 is a component of the nuclear envelope, the endo-plasmic reticulum, and the mitochondnial outer membrane.The localization of BcI-2 to the outer mitochondnial mem-brane was further substantiated using an in vitro importationassay. Our data are consistent with several other studiesusing immunofluorescence and/or electron microscopy (24-29) but disagree with a previous study, using subcellularfractionation and immunofluorescence staining, which con-cluded that BcI-2 resided exclusively within the inner mi-tochondnial membrane (10).

The failure of Hockenbery eta!. (10) to detect Bcb-2 in thenuclear membrane seems likely to be due to their having

assayed only whole nuclei. The major proportion of nuclearprotein is contributed by chromatin and nucleoplasm,which biases against detection of a protein confined to thenuclear envelope. Isolation of nuclear envelopes permittedus to detect Bcb-2 protein (Fig. 2, A and B). The relativeamount of BcI-2 in the nuclear membrane versus that of theouter mitochondnial membrane (Fig. 2A) seems likely to beunderestimated, because of the high proportion of envelopeprotein contributed by the pore complex and the nuclearlamina. The quantitation of immunogold labeling in the

electron micnographs probably resulted in a more accurateassessment of the proportion of Bcl-2 on the surface of eachofthese organdies: 34% in the nuclear envelope, 1 7% in theouter mitochondniab membrane, and 49% in other cytoplas-mic membranes including the endoplasmic reticulum (Table2). It is unclear why Bcl-2 was detected in the inner ratherthan the outer mitochondnial membrane in the previousstudy (1 0), but two other recent electron microscopic studiesof cell lines and of normal human lymphocytes have alsoconcluded that Bcl-2 is located in the outer membrane (27,29).

The location of an integral membrane protein on the sun-face of the nucleus, the endoplasmic reticulum, and mito-chondnia is to our knowledge an unprecedented situationand raises important questions about the nature of themechanism(s) directing the Bcl-2 protein to these diversemembranes. Bcl-2 lacks the classic signal sequences for tar-geting any one of these membranes, but it might belong toa class of proteins distributed to diverse intracellular boca-tions by virtue of COOH-terminal anchor sequences (34),since the COOH-terminal 25 amino acids are required forits insertion into microsomal vesicles (25) and also play animportant role in preventing apoptosis (26, 35). However,comparison ofthe Bcl-2 sequence with those of cytochnomeb5, UBC6, and KAR-1 , three tail-anchored proteins locatedin the endoplasmic reticulum and nuclear envelope, re-vealed no obvious targeting motif. Since it is widely ac-

cepted that the outer leaflet of the nuclear envelope sharessome proteins with the peninuclear endoplasmic reticulum(Ref. 36 and references therein), at least some of the Bcl-2protein detected in the microsomal fraction may have beenen route to the nuclear envelope. Relatively little is knownabout the mechanism for inserting proteins into the nuclearenvelope (37), but, in the case ofthe glycoprotein gp2l 0 andthe lamin B receptor, signals specifying location in thenuclear envelope have been defined in the transmembranedomain ofthe protein (36, 38, 39). Many proteins are trans-located into the interior ofthe mitochondnion by virtue of anamino-terminal amphipathic helix (reviewed in Ref. 40), butthe signals that direct proteins to the mitochondnial surfacehave not yet been defined. It is possible that there is more

than one mechanism which allows assembly into the mi-tochondnial outer membrane (30, 41). Perhaps Bcl-2 con-tains separate signals for binding to the endoplasmic reticu-bum, sorting to the nuclear envelope, and insertion into theouter mitochondnial membrane.

The biochemical mechanism of Bcl-2 action is unknown.Since it inhibits apoptosis in mutant fibroblasts lacking afunctional respiratory chain (28), and since the protein islocated on the outer aspect of the nuclear envelope, theendoplasmic reticulum, and the outer mitochondnial mem-brane, it presumably performs its function in the cytosol andnot inside mitochondnia, as previously suggested (1 0). A cell

1 2 3 1� �67

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7 :BcI-2 + pre OTC

Cell Growth & Differentiation 415

Table 2 Subcellular distribution of Bcl-2 in FDC-P1 cells infected with the hcl-2/neo virus, revealed by immunogold electron rn�roscopic analysis

This table summarizes the analysis fo more than 1 00 micrographs (92 844 points; se e also ‘ ‘Materials and Methods”).

Organelle Area (X0.01 pm2) Relative area %) Relative distribution” ‘V of total label

Nucleus

Mitochondria

Other cytoplasmic membranes (including endoplasmic reticulum)

27,71 1

8,323

56,810

30

9

61

10.6

1 7.9

7.7

34

1 7

49

,‘ Relatvie distribution is particles per relative area.

Fig. 4. Importation of Bcl-2 and preOTC proteins into isolated mitochon-

dna. In vitro translated [35Slmethionine-labeled Bcl-2 and preOTC proteinswere used in a standard mitochondrial importation assay (see “Materials and

Methods”). Proteins from fractions containing outer membranes or mitoplastsof untreated and Proteinase K-treated mitochondria were resolved by SDS-

polyacrylamide gel electrophoresis. Molecular weights are indicated in kDa

at left. The autoradiograph shown is representative of results obtained in 3independent assays.

normally needs both the intact nuclear genome and at leastone functional mitochondnial genome for optimal survival;all other organdIes and their components can be synthe-sized de novo. Given the established role of Bcl-2 in pro-tecting cells against apoptosis, a death process which cub-minates in DNA degradation, it might not be a coincidencethat Bcl-2 is localized on the outside of the two organdieswhich house DNA.

Materials and Methods

Isolation of Cell Lines. FDC-P1 myeboid cells (42), WEHI-231 B-lymphoma cells, B6.BW2 T-hybnidoma cells (43), andL929 fibrobbasts [subline LM(TK1] were infected with a ret-rovirus expressing a human bC!-2 cDNA and the neor gene(8). As a control, all four cell lines were also infected witha retrovirus expressing only the neor gene (provided by DrS. Cony). Infected cells were selected by culture in 1 mg/mbG418 (GIBCO, Gaithersburg, MD). Multiple independentclones of all four lines were generated by limiting dilutioncloning in 1 mg/mI G418 from separate pools of infect-ed cells. All cell lines were cultured in Dulbecco’s

modified Eagle’s medium supplemented with 50 �M

2-mercaptoethanob and 10% newborn calfserum and, in the

case of FDC-P1 cells, with -500 units/mI recombinantmouse IL-3 derived from the cell line X63/0 mIL-3 (44).

Immunofluorescence Analysis. Cells were resuspendedat 2 X 1 05/ml, centrifuged onto microscope slides, and fixedfor 3 mm in ice-cold acetone. Immunofluorescence stainingwas performed with the mouse monocbonal antibody Bcl-2-1 00 (45), which specifically recognizes human Bcl-2 pro-tein, followed by fluorescein isothiocyanate-conjugatedgoat anti-mouse immunogbobulin antibodies (Southern Bio-technology, Birmingham, AL). Human serum with a hightiter of autoantibodies against the pyruvate dehydrogenasecomplex E2 of mitochondnia from a patient with primarybiliary cirrhosis (46) was used as a control stain for an in-tramitochondniab membrane protein. In this case, we usedfluorescein isothiocyanate-conjugated sheep anti-humanimmunogbobulin antibodies (Silenus, Dandenong, Australia)

as the secondary reagent. Slides were viewed under a NikonOptiphot fluorescence microscope and photographed witha Nikon UFX-ll system, using Kodak ASA 2000 black andwhite film and exposure times between 1 and 10 s.

Subcellular Fractionation. Cells (-10�) in the exponen-tial phase of growth were harvested by centnifugation for 10mm at 1500 rpm in a GSA rotor (Sonvall). The cells werewashed in TKMS buffer [50 mxi Tnis-HCI (pH 7.5), 25 mx�KCI, 5 m� MgCb2, 250 m�a sucrose, 1 ms� dithiothreitol, and0.5 mtvt phenylmethylsulfonyl fluoride], resuspended in 4 mlof hypotonic buffer [10 mta 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 6.2), 10 mvi NaCI, 1 mxidithiothreitol, and 0.5 msa phenylmethybsulfonyl fluoride],and swollen for 1 5 mm on ice. The cells were transferred toa Dounce homogenizer and, after addition of 4 ml 2 x TKMSbuffer, homogenized with 1 5 strokes of a tight-fitting pestle.A crude nuclear fraction was collected by centnifugation for1 0 mm at 600 x g. Nuclei were purified from this pellet, andfractions containing nuclear envelopes and the porecomplex-bamina were prepared according to Genace et a!.(47). In order to increase the sensitivity of the immunobbot-ting assay for proteins located in the nuclear envelope, pu-nified nuclei were repeatedly subjected to digestion with

DNase I followed by centnifugation, to remove chromatinand nucleoplasmic proteins (47). Purified mitochondria

were prepared from the postnuclear supennatant and sub-fractionated into mitoplasts and mitochondnial outer mem-

branes, as previously described (48). Protein concentrationin subcellularfractions was determined using a BCA reactionkit (Pierce, Rockford, IL) and was confirmed by CoomassieBlue staining of SDS-polyacrylamide gels loaded with anabiquot of each fraction.

Marker Enzyme Assay. Enzymatic activity of monoamineoxidase, an outer mitochondnial membrane protein, and cy-tochnome coxidase, an inner mitochondnial membrane pro-tein, was measured as described (48, 49).

416 Intracellular Distribution of Bcl-2

Western Blotting. SDS-polyacnylamide gel electrophone-sis and Western blotting of subcellulan fractions wene per-formed as previously described (20) using monoclonalantibody Bcl-2-100. Analysis of the filters with aPhosphorlmager enabled quantitation of � 251-Pnotei nA-labeled bands.

Eledron Microscopy. Cells were prepared for immuno-gold labeling by the method of Pathak and Anderson (50).Cells were stained in four steps using (a) the mouse mono-clonal antibody BcI-2-1 00, (b) dinitrophenol-conjugatedgoat anti-mouse immunoglobulmn y antibodies (ICN Bio-chemistry, Sydney, Australia), (c) mouse monoclonal anti-dinitrophenol antibody (Oxford Biomedical, Oxford, Eng-land), and, finally, (d) 10 nm gold-labeled goat anti-mouseimmunoglobulin y antibodies (Amersharn, Amensham, Eng-land). Samples were viewed on a Philips CM1 2 transmissionelectron microscope. Micnognaphs were printed at a finalmagnification of X 51,000.

Statistical analysis of Bcl-2 localization detected by im-munogold electron microscopy was performed by point-h it-counting. An orthogonal grid on transparent plastic (areaassociated with each grid point equal to 0.01 pm2) was oven-layed on each micrognaph, and the number of points oven-lying the nucleus, mitochondnia, and the remaining cyto-plasm was determined, together with the number ofimmunogold particles associated with these compartments.In addition, the number of particles associated with mem-branes was recorded. Data were analyzed using a nonpara-metric two-factor analysis of variance (an extension of theKruskal-Wallis test). A total of 92,844 experimental and

78,31 3 control points were analyzed for FDC-P1 myeloidcells infected with either the bcl-2/neo or the control neonetnovirus.

Importation of Bcl-2 into Isolated Mitochondria. Theplasmid pBluebc/-2 was constructed by inserting the EcoRI-Taqlfnagmentofhuman bcl-2 cDNA(51) into pBluescnipt KS(Stratagene, San Diego, CA), cut with EcoRI and C/al. Afterlinearizing pBluebc/-2 with KpnI, BcI-2 protein was tnans-lated in vitro using FLEXI lysate (Promega, Madison, WI) andimported into mitochondnia isolated from rat liver, as pre-viously described (31). All importation assays were carriedoutfon 30 mm at 30#{176}C.Mitochondnia (2.5 mg mitochondnialprotein) were subsequently purified and fractionated intoouter membranes and mitoplasts using established pnoce-dunes (32, 48). Importation ofthe in vitrotranslated pneOTC,using the plasmid cOTC1 4, served as an internal control ofthe importation assay (31).

Acknowledgments

The authors acknowledge Drs. S. Cory, A. W. Harris, J. M. Adams, P. Hoj, and

N. J. Hoogenraad for their support, helpful advice, and stimulating discussionsand thank Drs. S. Cory, N. Kronidou, and I. Leighton for critically reviewingthe manuscript. Expert technical assistance was provided by M. Stanley, J.Beaumont, and L. Gibson. We also thank Drs. S. Cory and D. Vaux for neo

and bc/-2/neo virus-producing cell lines, Dr. D. Y. Mason for the Bcl-2-1 00monoclonal antibody, Dr. S. Whittingham for antiserum from a patient withprimary biliary cirrhosis, Dr. N. J. Hoogennaad for the cOTC14 plasmid, and

Drs. H. Karasuyama and F. Melchers for the recombinant mouse IL-3 pro-

ducing cell line X63/0 mIL-3.

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