THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267, , No. Issue of October 15, pp. 21072-21079.1992 Printed in U. S A .

Purification, Functional Characterization, and cDNA Sequencing of Mitochondrial Porin from Dictyostelium discoideum”

(Received for publication, May 11, 1992)

Heike Troll$, Dieter MalchowS, Annette Muller-Taubenbergerg, Bruno Humbelg, Friedrich Lottspeicht, Maria Eckeg, Gunther Gerischg, Angela Schmidl, and Roland Benzli From the SFakultat fur Biologie, Universitat Konstanz, 0-7750 Konstanz, §Max-Plnnck-Institut fur Biochemie, 0-8033 Martinsried, and the YLehrstuhl fur Biotechnologie, Biozentrum der Uniuersitat Wurzburg, 0-8700 Wurzburg, Federal Republic of Germany

Porin of Dictyostelium discoideum was extracted from mitochondria with Genapol X-80 and was puri- fied by hydroxyapatite and CM-cellulose chromato- graphy. The purified protein displayed a single band of 30 kDa in SDS-polyacrylamide gel electrophoresis. The formation of channels in artificial lipid bilayer membranes defined its function as a channel-forming component. Its average single-channel conductance was 3.9 nanosiemens in 1 M KCl, which suggested that the effective diameter of the channel is approximately 1.7 nm at small transmembrane potentials. The chan- nel displayed a characteristic voltage dependence for potentials higher than 20 mV. It switched to substates of smaller conductance and a selectivity different to that of the open state. The closed state was stabilized at low ionic strength. The cDNA sequence of mitochon- drial porin from D. diseoideum was determined. It showed little sequence similarities to other known mi- tochondrial porins. The functional similarity, how- ever, was striking. Localization of the porin in the mitochondrial outer membrane was confirmed by im- munogold labeling of cryosections of fixed cells.

During oxidative phosphorylation, many substances, mostly anionic ones, have to pass mitochondrial inner and outer membranes. The outer membrane acts as a molecular filter with defined exclusion limits for hydrophilic mitochondrial substrates (1). The component active in molecular sieving is porin (2, 3), a major outer membrane protein, also known as voltage-dependent anion-selective channel (VDAC) (4, 5). The name porin was chosen in analogy to the pore-forming proteins in the outer membrane of Gram-negative bacteria (6). Structural and functional similarities suggest a conge- niality, since both bacterial and mitochondrial porins form p- barrel structures with probably 16 &strands crossing the membrane (7-9).

The isolation and characterization of mitochondrial porins from different eukaryotic organisms made a detailed analysis of the biochemical and biophysical properties of the channels possible (2-5, 10-19). The molecular mass of mitochondrial porins varies between 29 and 35 kDa in different organisms.

schaft Grants SFB 156, SFB 176/B9, and SFB 266/D7 and by the * This work has been supported by Deutsche Forschungsgemein-

Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

to the GenBankTM/EMBL Data Bank with accession number($ The nucleotide sequence(s) reported in thispaper has been submitted

M96668.

Mitochondrial porins from Saccharomyces cereulsiae (20), Neurospora crassa (7), and a human B-lymphocyte cell line (21) have been sequenced on the DNA or protein level. These porins show little similarities through their entire sequences, they contain predominantly hydrophilic regions with many charged residues and have isoelectric points between pH 7 and 8 (12, 22). All mitochondrial porins studied so far are voltage-dependent and are in the open state at small voltages and switch to closed states at voltages above 20-30 mV (1-5, 13-17). Measurements of the ion selectivity revealed weak anion selectivity of the open state of eukaryotic porins (1, 13, 14), whereas the closed state is cation-selective (23). This suggests charged groups oriented to the lumen of the pore. The channel-forming unit is probably composed of one or two polypeptide subunits, associated with sterols and lipids (24- 26). It forms a cylinder with an outer diameter of about 5 nm and an inner diameter of 1.8-2.5 nm (26-28), which prevents the passage of molecules larger than 4-5 kDa.

The different selectivities of mitochondrial porins in the open and closed states suggest that the channel is involved in the regulation of mitochondrial metabolism (29). Mitochon- dria in situ have various contact sites between the inner and outer membrane, depending on their energetic state (30). These contact sites may be responsible for electric coupling of the two membranes and thus for the control of outer membrane permeability. Furthermore, hexokinase and glycer- okinase bind to mitochondrial porin from the cytoplasmic side. These enzymes couple oxidative phosphorylation and glycolysis, and binding to porin increases their activity (31).

In this paper we describe the isolation and purification of mitochondrial porin from Dictyostelium discoideum, a strictly aerobic microorganism with tubular mitochondria, which is neither directly related to fungi nor to higher eukaryotes. The purified protein was reconstituted in lipid bilayer membranes, and its electrophysical properties were studied. A monoclonal antibody against the protein allowed the isolation of cDNA clones from expression libraries. Surprisingly, the primary sequence of Dictyostelium porin exhibited only limited simi- larity to the known sequences of mitochondrial porins, al- though its function as a channel-forming component was almost indistinguishable from that of other mitochondrial porins.

MATERIALS AND METHODS

Growth of Dictyostelium Cells and Isolation of Mitochondria-D. discoideum Ax2 cells were grown in liquid culture media (32) on a shaker (150 rpm) at 23 “C up to a density of about lo7 cells/ml. The cells were washed twice with ice-cold Sorensen-phosphate buffer (13 mM KH2P04, 4 mM Na2HP04, pH 6.0) and resuspended in buffer s (200 mM sucrose, 20 mM Tris-HC1, pH 8.0,l mM EGTA, 0.2% bovine serum albumin). Cells were then disrupted in a Waring blender at

21072

Mitochondrial Porin from D. discoideum 21073

4 “C for 25 s on position “low” followed by 5 s on position “high.” Mitochondria were enriched by centrifugation at 4 “C for 20 min at 27,000 X g (32). The pellet was resuspended in the same volume of buffer S and recentrifuged for 5 min at 400 X g to remove unbroken cells. The supernatant was centrifuged again for 10 min at 16,000 X g to give the final mitochondrial pellet. Alternatively, the cells were disrupted in the cold in a Parr bomb at 1000 psi., in the absence of bovine serum albumin. The 16,000 X g pellet was centrifuged for 1 h at 100,000 x g in 30% Percoll, and the mitochondria-containing layer was either used fresh or frozen.

Purification of Dictyostelium Porin-The porin was isolated from mitochondria essentially as described elsewhere (13, 33). The mito- chondrial pellet (about 60 mg of total protein, 2 ml) was suspended in cold buffer D (10 mM HEPES, pH 6.8, 1 mM EDTA) to a final protein concentration of 30 mg/ml and centrifuged for 30 min at 100,000 X g at 4 “C. The pellet was resuspended for 30 min in 7 ml of buffer D supplemented with Genapol X-80 (final detergent concen- tration 2.4 mg of Genapol X-SO/mg of protein) and centrifuged for 30 min at 100,000 X g. The extract was applied to a HTP’ column preequilibrated with buffer D, and porin was eluted with the same buffer supplemented with 100 mM KCl. After 12-h dialysis against buffer E (10 mM MES, pH 6.0, 1% Genapol X-80), the porin- containing fractions were applied to a CM-cellulose column. The porin was eluted with a linear salt gradient of 0-200 mM NaCl in buffer E. Protein was determined according to Ref. 34. The fractions were assayed in parallel for porin activity and for reactivity with monoclonal antibody 100.

SDS-PAGE and Immunoblotting-Purity and relative molecular mass of the Dictyosteliurn porin was examined by 12% SDS-PAGE. To remove detergent, the protein was precipitated according to Wes- sel and Flugge (35). The resulting pellet was dissolved in 10-20 pl of sample buffer (containing 0.5 M Tris-HC1, 10% SDS, 400 mM EDTA, 4% mercaptoethanol) and incubated for 5 min at 37 “C and for 3 min at 100 “C. The gels were stained with Coomassie Blue. For immuno- blotting, proteins were separated by SDS-PAGE in 12% gels, trans- ferred onto Schleicher and Schuell BA85 nitrocellulose filters, incu- bated with hybridoma culture supernatant, and labeled with alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson Immuno Re- search Laboratories) using standard procedures. Blots were scanned in the remission mode using an Elscript 400 AT scanner (Analysen- technik Hirschmann, Taufkirchen, Federal Republic of Germany).

Black Lipid Bilayer Membrane Experiments-Reconstitution of porin into artificial lipid bilayer membranes has been described previously (36). Membranes were formed from a 1% (w/v) solution of diphytanoyl phosphatidylcholine (Avanti Polar Lipids, Birming- ham, AL) in n-decane in a Teflon cell consisting of two aqueous compartments connected by a circular hole. The area of the hole was 0.2 mm2 for single channel experiments and 2 mm2 for macroscopic conductance measurements. Membranes were formed across the hole and 10-100 ng of porin was added to 5 ml of the aqueous phase at one side of the membrane. The aqueous salt solutions (analytical grade, Merck, Darmstadt, FRG) were used unbuffered and had a pH around 6.0. The temperature was kept at 25 “C throughout.

The membrane current was measured with a pair of calomel electrodes switched in series with a voltage source and an electrometer (Keithley 602). For single-channel recordings, the electrometer was replaced by a current amplifier (Keithley 427). Zero-current mem- brane potential measurements were performed by establishing a salt gradient across membranes containing 100-1000 porin channels as has been described earlier (37).

cDNA Cloning and Sequencing-A X g t l l cDNA library from starved D. discoideum cells, kindly provided by Dr. Richard Kessin, Columbia University, was screened with mAb 100 and 1251-sheep anti- mouse IgG (Amersham). EcoRI fragments of a positive clone, Xcdp100, were subcloned into pUC19 and sequenced by the chain termination method (38,39) using uni, reverse, and sequence-specific oligonucleotide primers. The orientation of the EcoRI fragments was determined after amplifying the entire X clone by polymerase chain reaction and sequencing the region comprising EcoRI sites. Because the clone lacked the 5’-end of the coding region, clone Xcdp83 was isolated from a h e l l cDNA library of growth-phase cells (library courtesy of Dr. Arnold Kaplan, Saint Louis University), by the use of the 5”EcoRI fragment of XcdplOO as a probe. Clone Xcdp83 from this library was partially sequenced in order to obtain the 5’-coding

The abbreviations used are: HTP, hydroxyapatite; PAGE, poly- acrylamide gel electrophoresis; MES, 2-(N-morpholino)ethanesul- fonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; S, siemens.

and -flanking sequence missing in Xcdp100. For Northern blots, RNA was prepared by phenol/chloroform

extraction from growing cells and from developing cells harvested at 5 h of starvation. After electrophoresis in 1.2% agarose-formaldehyde gels, nucleic acids were transferred to nitrocellulose filters (BA85, Schleicher and Schuell) and were hybridized with EcoRI cDNA fragments derived from clone hcdp100. Labeling and detection reac- tions were performed using the ECL system (Amersham) under standard conditions. The sequences were analyzed using programs from the University of Wisconsin Genetics Computer Group (40).

Cyanogen Bromide Cleavage and Protein Sequencing-Purified po- rin was incubated for 4 h at room temperature with 10% (w/v) cyanogen bromide in 70% formic acid. The sample was 6-7-fold concentrated by evaporation and subjected to amino acid sequence analysis. Uncleaved or cleaved porin was spotted onto a siliconized glass fiber disk (Glassybond, Biometra, Gottingen, FRG), dried, ex- tensively washed with water, and sequenced in an Applied Biosystems 477A Sequencer equipped with a 120A phenylthiohydantoin analyzer according to the instructions of the manufacturer.

Immumlabeling of Fixed Cells and Cryosectiom-Monoclonal an- tibodies were raised against the detergent-insoluble fraction from D. discoideum strain AX2 cells. The cells were extracted with 1% Triton X-100 and injected into BALB/c mice using Alugel S (Serva, Heidel- berg, FRG) as an adjuvant. Spleen cells were fused with Sp2/01 myeloma cells, and antibody 70-100-1, designated in this paper as mAb 100, was identified in hybridoma culture supernatant by im- munoblotting.

For immunolabeling, cells were fixed for 30 min at room tempera- ture in a mixture of 15 ml of a saturated aqueous solution of picric acid and 85 ml of 10 mM PIPES buffer, pH 6.0, containing 2 g of paraformaldehyde (41). For fluorescence labeling, cells were post- fixed with 70% ethanol, washed again in the buffer, incubated, after washing in the buffer, with mAb 100-containing hybridoma culture supernatant and subsequently with fluorescein isothiocyanate-con- jugated goat anti-mouse IgG antibodies (Jackson Immunoresearch Lab, Inc., West Grove, PA). For immunogold labeling of sections, cells fixed with picric acid/formaldehyde were embedded in gelatin, cubes of about 1 mm3 were cut and infiltrated with 2.3 M sucrose for cryosectioning according to Tokuyasu (42). Sections obtained with a Reichert-Jung FC4D Ultracut E were labeled with mAb 100 using hybridoma culture supernatant. The monoclonal antibody was de- tected by goat anti-mouse antibodies conjugated to l-nm colloidal gold (Janssen Life Sciences, Olen, Belgium). The ultra-small gold particles were visualized by silver deposition according to Danscher (43) following the procedure of Stierhof et al. (44). The solutions of fluorescein isothiocyanate- or gold-conjugated secpndary antibodies were diluted 1: lOO into phosphate-buffered saline containing 0.05% fish gelatin (45) and 0.5% bovine serum albumin to mask unspecific protein binding sites (46, 47).

RESULTS

Purification of Dictyostelium Porin and Reconstitution Ex- periments-For the extraction of porin from Dictyostelium mitochondria, Genapol X-80 was used because this detergent solubilizes outer mitochondrial proteins to a higher extent than inner membrane proteins. Porin was purified from the extract by chromatography on HTP and CM-cellulose col- umns. In contrast to other mitochondrial porins (13, 16, 17), only minor amounts of Dictyostelium porin were found in the eluate of the HTP-column just after the void volume, and 100 mM NaCl had to be added to buffer D to eluate porin contam- inated by minor amounts of other proteins. Final porin puri- fication was achieved by chromatography on CM-cellulose. Dictyostelium porin bound to this material at pH 6.0 and was eluted from the column with 35-80 mM NaCl (Fig. 1). SDS- PAGE of the protein showed a single band with an apparent molecular mass of 30 kDa (see inset of Fig. 1).

When the Dictyostelium porin was added in quantities of 10-100 ng/ml to the aqueous phase on one or both sides of a lipid bilayer membrane, the conductance of the membrane increased by several orders of magnitude. The time course of increase was similar to that described previously for other mitochondrial or bacterial porins (1, 3, 11). After a rapid

21074 Mitochondrial Porin from D. discoideum

a OI 1000 2oo f 1 r

*0° 0 ~: 0 5 1 0 1 5 2 0 2 5

fraction number

FIG. 1. CM-cellulose chromatography and SDS-PAGE of Dictyosteliurn porin. The porin-containing fractions of an HTI' column were applied to a CM-cellulose column and porin was eluted with a linear NaCl gradient (0-200 mM, trianglrs). The minor protein peak eluting in fractions 14-20 contained the purified protein. The fraction volume was 4 ml. 1 1 pg of the protein from fraction 16 were applied to 12% SDS-PAGE and migrated as a single band with an apparent molecular mass of ahout 30 kDa. The gel was stained with Coomassie Blue. The posit inns of molecular mass markers are indi- cated.

J

I 1

10nS lOOpA

- lm in

FIG. 2. Stepwise increase of the membrane current after the addition of the Dictyostelium porin to the aqueous phase bathing a black membrane. The memhrane was formed from 1"; diphvttlnoyl phosphatitlvlcholine/n-decane. The aqueous phase con- tained 2 ng/ml porin and 1 M KCI, pH 6. The applied voltage was 10 mV; the current prior to the addition of the protein was less than 0.5 pA; 7: 25 "C.

increase during 15-20 min, the membrane conductance in- creased at a much slower rate. Addition of the detergent Genapol X-80 alone in control experiments did not lead to any appreciable increase in the membrane conductance.

Single-channel Experiments-The addition of Dictyoste- lium porin a t very small concentrations (5 ng/ml) to the aqueous solution on one side of a lipid bilayer membrane allowed the resolution of step increases in conductance; each step corresponded to the incorporation of one channel-form- ing unit into the membrane (Fig. 2). Most of the conductance steps corresponded to a conductance of 4 nS in 1 M KC1 (see the histogram of Fig. 3), and only a small number of steps to about 2.5 nS. These smaller channels have been observed together with the 4-nS channels for most mitochondrial po- rins (1). Under the low voltage conditions of Fig. 2, most of the steps were directed upwards, which indicate open states

P (GI

0.4

0.2

T

1 J 0 2 4 6 G I n S

FIG. 3. Histogram of conductance fluctuations observed with membranes from diphytmoyl phosphatidylcholinc/n-de- cane in the presence of Dictyonteliurn parin. The aqueous phnse contained 1 M K(:l. The applied voltage was I O m y . The mean value of all upward directed steps wns 3.9 nS for 238 single events: 7'. 25 -('.

TABLE I Aurragr sin&-chnnnrl conductance of I)ictyostrlium pnrin in

diffcwnt s d t so/ulions

The solutions contained 5-10 ng/ml I>icl.vosfr/ium porin and less than 0.1 pg/ml Genapol 5-80; the pH was hetween 6.0 and 7.0. The membranes were made of diphytanoyl phosphatid~lcholine/n-tlecRnc: 7' = 25 "C; V,,, = 1 0 mV. (; represents the average nf at least 50 conductance steps. c is the concentration of the salt solution and n is its snecific conductance.

Salt c (; (;/I7

M n S I O * crn

KC1 0.01 0.048 3.4 0.03 0.14 3 . 3 0.1 0.44 3.1 0.3 1.5 1

3.7 3.9 3.5

3 11 KHr 1 4.0 3.6

3.9

NaCI 1 3.7 4.4 LiCl 1 3.4 4.R MgClz 0.5 3 . 3 Tris-CI 0.5 1 .'i 5.3

5 . 2

Tris-HEPES 0.5 0.022 3.1 K-MES 0.5 0.7.5 K-acetate 1 1.6 2.2

2 5

of the pore. Only a few downward steps were observed. At higher transmembrane potentials the closing events became more frequent (see below).

Dictyostelium porin was permeable for a wide variety of ions (Table I). Although the channel conductance was influ- enced considerably by the different salts and concentrations, the ratio between single-channel conductance C and the bulk aqueous conductivity varied only by about a factor of 2, i . ~ . the ions seemed to move within the pore as in the aqueous environment. This finding suggests that the Dictyosteliurn porin forms a wide water-filled channel. Nevertheless, the data of Table I show a certain extent of ion selectivity. For instance, the difference of the single channel conductances between potassium acetate and LiCl suggests that the porin is anion-selective; the exchange of K' by the less mobile Li' (3.9 uersus 3.4 nS) had a smaller effect on the single-channel conductance than replacement of CI- by the less mobile acetate anion (3.9 uersu.7 1.6 nS). In this respect it has to he noted that K' has the same aqueous mobility as CI- (both

Mitochondrial Porin from D. discoideum 21075

about 70 mS/(cm X M)) and that Li+ has the same one as acetate (both about 40 mS/(cm X M) (48).

Zero-current Membrane Potentials-Zero-current mem- brane potentials were measured to study the ion selectivity of Dictyostelium porin in detail. Membranes were formed in 10 mM KC1, and porin was added to the aqueous phase when the membranes were in the black state. After incorporation of 100-1000 channels into a membrane, salt gradients were established by addition of small amounts of concentrated KC1 solution to one side of the membrane.

Unexpectedly, the more diluted side of the membrane be- came positive, which indicates preferential movement of K+ ions. From the single-channel data, preferential movement of C1- was predicted (see above). Very small gradients (c”/c’ = 1.5) resulted in a positive zero-current potential (see Fig. 4). However, when the experiments were repeated starting from 50 mM KC1 (instead of 10 mM), the more diluted side of the membrane became negative (see Fig. 4). These results indicate that the ionic strength of the aqueous salt solution influences the ion selectivity of Dictyostelium porin. At a salt concentra- tion of 10 mM the channels are in the cation-selective “closed” configuration, whereas they are anion-selective and in the “open” state at 50 mM ionic strength. The zero-current mem- brane potentials were analyzed using the Goldman-Hodgkin- Katz equation (37). The ratio of the permeabilities, Panion/ Pcation, was 2.0 at 50 mM KC1 and 0.1 at 10 mM KC1. No similar influence of the ionic strength on the selectivity of a channel has been reported before.

Voltage Dependence-The results described above sug- gested a voltage dependence for Dictyostelium porin. To con- firm this, appropriate experiments were performed under multichannel conditions, i.e. many pores were reconstituted into the membrane. The voltage across the membrane was set to different potentials ranging from 10 to 100 mV, and the membrane currents were monitored on an oscilloscope screen. Immediately after application of the voltage, the current was a linear function of the applied membrane potential. For voltages larger than 20 mV, the membrane current decayed thereafter exponentially to smaller values (see Fig. 5 A ) . The maximal decrease of conductance was observed at a voltage of 80-100 mV. At this potential, the conductance G was about

‘4, I mV 60

30

0

-30 1 2 5 10 20

c”/c’

FIG. 4. Zero-current membrane potentials V,,, of mem- branes formed from diphytanoyl phosphatidylcholine/n-de- cane in the presence of Dictyostelium porin as a function of the KC1 gradient c”/c’ across the membrane. The potential corresponds to the more dilute side (c’) of the membrane. The lines were drawn according to the Goldman-Hodgkin-Katz equation (37) with the indicated values for PJP.. Note that the potential was positive on the more dilute side for c’ = 10 mM (circles) and negative for c’ = 50 mM (triangles). T, 25 “C.

A

I

-00 -40 0 40 BO ym I m~

FIG. 5. A, relaxation of the membrane current in the presence of Dictyosteliurn porin. The membrane potential was rapidly taken from 0 to 10 mV (curue I ) , from 0 to 50 mV (curue 2) , and from 0 to 100 mV (curue 3, same membrane). The membrane was formed from diphytanoyl phosphatidylcholine/n-decane. The aqueous phase con- tained 1 M KC1 and 20 ng/ml porin; T = 25 “C. 23, bell-shaped curve for C/Co measured as a function of the transmembrane potential at the same membrane. Analysis of the data with Equation 1 suggested that the number n of gating charges moving through the entire membrane potential was 2 and that the potential, Vo, a t which half of the channels are closed was 52 mV.

40-50% of the initial value Go. Higher potentials did not cause a further conductance decrease. Fig. 5B shows the ratio G/Go as a function of the transmembrane potential V,.

The number of channels in the open state, No, divided by the number of channels in the closed configuration, N,, are given by the following equation (4),

N,/N, = exp (nF(V, - V d / R r ) (1)

where F, R, and T are standard symbols, n is the number of charges moving through the whole membrane (or the entire electric field) for channel gating, and V,, corresponds to the potential at which half of the channels are closed. The semi- logarithmic plot of the ratio NJN, as a function of the transmembrane potential V,,, could be fitted to a straight line with a slope of 12 mV for an e-fold change of NOIN,. From this result n = 2.0 is calculated for Dictyostelium porin. This means that approximately two charges move through the entire membrane for channel gating. A similar number of gating charges have been found for other mitochondrial porins (1, 13), with the exception of porin from rat brain (n = 1 (15)).

Screening for an Anti-porin Monoclonal Antibody-Mito- chondria are known to be attached to the cytoskeleton, and extraction of porin by Triton X-100 has been shown to be

21076 Mitochondrial Porin from D. diacoideum

reduced by binding to MAP2 (49). Antibodies were raised in mice against the Triton X-100-insoluble fraction of Dictyos- telium cells, which contains mitochondria within a network of cytoskeletal filaments. In immunoblots of total cellular proteins separated by SDS-PAGE, one of these antibodies, mAb 100, labeled specifically a protein of 30 kDa, the size of porin. When used for immunofluorescent antigen localization, mAb 100 labeled mitochondria exclusively (Fig. 6A) . The label was restricted to the periphery of the organelles, as indicated by rings of fluorescence surrounding the mitochondria, which appear as dark dots in phase-contrast images (Fig. 6R). Col- ocalization of the antibody label with the outer mitochondrial membrane was confirmed on the electron microscopic level by immunogold labeling of cryosections. Neither the mito- chondrial matrix nor the tubuli extending from the inner

B "

C

FIG. 6. Immunolabeling of whole cells ( A and B ) and of cryosections (C) with mAb 100. For A and H , growth-phase cells spread on a glass coverslip were fixed and fluorescent laheled. The label shown in A is localized to the periphery of mitochondria which in the phase-contrast image of R appear as dark dots. For C, cryosec- tions were immunogold-laheled. The gold particles are concentrated along the outline of mitochondria. The nucleus (n), including its cap ( c ) , the equivalent of a nucleolus (55), are unlaheled. Hnrs indicate 10 pm for A and H , and 0.5 pm for C.

membrane of D. discoideum mitochondria were labeled (Fig. SC).

The protein recognized by mAb 100 cofractionated on a CM-cellulose column with the channel-forming acitvity of porin (Fig. 7). Since this protein had also the predicted size and localization of porin, mAb 100 was employed to the screening of expression libraries in order to isolate cDNA clones for sequencing.

Cloning and Sequencing of Dictyostelium Porin-Two dif- ferent Xgtll expression libraries were screened for porin se- quences as described under "Materials and Methods," and one clone from each library was used for sequencing. In one of these clones, Xcdp100, the coding region appeared to be complete except for the li'-end. The Fj'-coding and -flanking region was sequenced in the second clone, XcclpR3. Northern blots were probed separately with the three EcoRI fragments of clone Xcdp100. Each suhclone recognized a single transcript of 1.1 kilobases. This transcript was present in growing cells as well as in cells harvested a t 5 h of starvation, in accord with the continued presence of the protein during early de- velopment.

The complete cDNA and deduced amino acid sequence is shown in Fig. 8. It indicates a protein of 30.2 kDa. To confirm

A

30 b U Da

1 2 3 4 5 E 7 8 9 10 11 12 1 3 1 4 1 5 16

fraclion number

I d \

I -c- - 9 0 2L 1 t o 4

6. :I 1 . .. . p o l l e d f r a C t l O n %

FIG. 7. Comparison of antibody binding and conductivity in fractions of the final porin purification step. Frnctinn.; of a ('\I- cellulose column were subjected t o SI>S-I'A(;F. and immunohlotted with mAh 100 ( A ) . The immunolahel was quantified hy scanning as described under "Materials and Methods" ( H 1. Pore-forming activitv was measured in three pools. containing traces. high, and low amounts, respectively, ofthe antihody-recognized 3 O - k I h protein (( '). 15 pl of these pools were added to 5 ml of 1 M KC1 solution. and the specific conductance of diphvtanovl phosphatidvlcholin~/n-drcnne memhranes was measured at 'LO min after addit ion of the protein; C:, = 20 mV, T = 25 "C.

Mitochondrial Porin from D. discoideum 21077

- 2 5 A A A 6 C 1 1 l l A A l A 1 l A A T A A A C A A A

A l G A A C C C A G G l C l C l A C 6 C C G I ~ l l A A C C A A A C C A A C l G C ~ G A l l ~ C A ~ C A A A A A G 6 A ~ H I P G l l A D L T K P l A O F l K K O 20 ........... f A t l i K L O T l i K G K l 6 S I V A 10 l l C G C A G A A A C C I T C A A A C l C G A l A C C A C C ~ l C A A A G 6 C A A A l A l 6 6 l l C A A ~ l G ~ l 6 C A

G 1 C A C T G A l A l l A A A G A C A S l G G ~ G l l 6 l l G C C ~ C A A l ~ C A A C C A A A A 6 C l G A l ~ l C A C C V I D I K O S G V V A S I O P K A D f T 6 0

A A A I A T C l C 6 6 1 A A A 6 T C l C A A A l 6 G ~ A A C l ~ l A C ~ 6 ~ C 6 A ~ A C C A A ~ G 6 ~ G ~ A A A 6 A A A K Y L 6 K V S N 6 N i l V D I W G V X K 110

V G G I G A A I l C A C C A l I G A A A A T I T T I T T t t L 6 6 T T T l ) i l l l A A A A 6 C C 6 l C G C C A A l 6 G ~ G A l l C A G t i l l t N I I P G L K A V A N G D S 100

V L A A C A A A A I I I C l C A A C C G A A l l C C A A l A l A A A A A A 6 A l A A A L ~ ~ 6 C l ~ l C A C C C ~ ~ ~ ~ ~ K O N i S l t l O l X K D K I A i T L i 120

~ ~ T C A C A A C A A ~ A A A I C C T T C A A ~ A C ~ ~ C A ~ ~ A ~ C ~ ~ ~ C C ~ C A ~ ~ A A C C C A A C ~ ~ ~ C ~ C ~ ~ H ~ N K S ~ N T S L A ~ L I N P T ~ S 160

G 1 1 G 6 1 6 l C C L A G C l G A A G G T I I I S ( C I L I I I 1 I C L I T C L C A ~ C V G V O I L G N A X I T L L N V N A I I I 6 0

A C C I I C A 6 A C C A C G T C C A G A C G l C l l l G ~ l l C A A l C G l C 6 A ~ A 6 A l l C A ~ 6 G A ~ A A A C A A 1 I R P R P O V T V S I V O R i H D K O I110 ... A i C C l C T T A l C C A C C C l C T A C A C l G C C A C C l C A A A A l l A ~ C C l ~ l G C l G G ~ 6 A ~ 6 l l A C l . . . . . 0 . . I L L S l L I T A 1 S K L S i A G D V T 200

v ~ ~ C G A C ~ ~ C A A A G C C ~ C ~ ~ A A A A A G C A C ~ A ~ C A ~ ~ C A A C G ~ ~ G ~ ~ A C C C A A ~ A C A A A A ~ C V D L K A S ~ K A P S I H V G T O Y K I 220

G A l l C C G C C A G 1 C T C C T C A A A G C l A A I 6 ~ ~ A A C A A C A A C A G A A A A G ~ l A L C A l C ~ C l I ~ C D S A S L L K A K V N N N R K V N I S I 210

A l C I A C A ~ l A C T A G C A A C A A C I C l A A A l l C G l l l l A 6 G l ~ 6 G L A l G ~ C A A ~ A C l A A A A A C I I N T S N N T I F V ~ G Y H V N T K N 2 6 0

l T I A A A C A A G G C A A l A C l l T l G G l G C l A C l G l l A A C l ~ A A C l C l l l L A A ~ l 6 ~ A A A C l l ~ I K O G N I F G A T V N L I L ' 271

l A l l l A C A l T i l l A A A ~ A G A L A A A L A A A A A A C A A A A A ~ A C A C C A l A A I A A l A l A A A A A A A

A A A l A A l A A l I 6 l A A A C A A l l G l A l A A l l ~ l C A A A l l l G G l A A A A l C A A l l l ~ l l ~ C A l l

l l l A C l A L A l A I l l G 6 ~ A A A A A G A A l l l l ~ ~ A A A A A A A A A A A A G l A A l A A

l G l A l l l l A l l l l T l l T l l l l A A A A A C l l C l A A A A A A A A I A A A A A A A A A A A A A A A A A A L A

A A A A A A A A A A A A A A A A A A A A A A A 1103

0 200 I I

3 1 r

hydrophobic

hydrophilic

I I

0 200

FIG. 8. Combined cDNA sequence of the coding and flank- ing regions of clones XcdplOO and Xcdp83 and derived se- quence of the protein ( A ) and hydropathy plot (23). Clone XcdplOO was completely sequenced in both directions and comprised nucleotides +6 to +1103. A EcoRIIKpnI fragment from clone hcdp83 containing nucleotides -25 to +650 was partially sequenced, the region between nucleotides -25 and +87 in both directions. EcoRI and KpnI cleavage sites are indicated by open and closed triangles, respectively. Polyadenylation signals are underlined. Two stretches of the amino acid sequence determined by Edman degradation after cyanogen bromide cleavage of the Dictyosteliurn porin are indicated by solid dots. (The leucine residue in position 186 was not unambig- uously identified by protein sequencing).

that this sequence corresponds to the protein recognized by mAb 100, the 30-kDa protein labeled by the antibody was purified and subjected to amino acid sequence analysis. No sequence could be obtained, indicating a blocked N terminus. Therefore, the protein was cleaved by cyanogen bromide. Since the predicted amino acid sequence contains only one internal methionine residue, the cleavage mixture was se- quenced directly without separation of the fragments. Two

sequences were obtained which correspond to the marked portions in Fig. 8.

DISCUSSION

We have isolated a channel-forming protein from Dictyos- telium mitochondria which has an apparent molecular mass of 30 kDa on SDS-PAGE (see Fig. 1). This is very close to the molecular mass calculated from the cDNA-derived amino acid sequence, 30.2 kDa, and to the masses of mitochondrial porins from other organisms, which range from 30 to 35 kDa (1, 13, 16, 17). In its chromatographic behavior the Dictyos- telium porin is distinguished by the binding to hydroxyapatite and CM-cellulose under conditions where other porins do not bind. The binding to hydroxyapatite in the presence of deter- gent suggests that portions of the polypeptide chain are ex- posed on the surface of detergent micelles that are larger in Dictyostelium porin than in other porins (3, 12, 13, 18, 50). The binding to CM-cellulose may be related to the high PI of about 10 as calculated from the sequence of Dictyostelium porin.

In Fig. 9 the amino acid sequence of human (21) and the cDNA-derived sequences of N . crassa (7) and S. cerevisiae (20) porins are compared with the porin sequence from D. discoideum. These sequences are related to each other, al- though only 14 (out of about 280) amino acid residues are strictly conserved in all four porins (marked by dots in Fig. 9). The most outstanding identity is the Gly-Leu-Lys triplet near amino acid 100. No such identities have been found in sequences of porins from phototrophic and other bacteria, although the functions of these porins as channels are similar to those of the mitochondrial porins. The hydropathicity plot indicates that the Dictyostelium porin is predominately hy- drophilic (Fig. 8B) and clearly distinguishes its channels from the ion channels in neurons and muscle fibers with their typical &-helical structure (51-53). Porins are encoded in the nucleus; the amphiphilic N-terminal region of the Dictyoste- lium porin may be involved in target recognition (7, 20).

As compared with the three other mitochondrial porins, the sequence of the Dictyostelium porin is less well conserved. Particularly in the middle region of the Dictyostelium se- quence there are long stretches which show no identities to the other three sequences. These results suggest that the @- barrel structure of the membrane channels, as it has been resolved in a bacterial porin by x-ray diffraction (8), tolerates extensive amino acid variations without substantial altera- tions in the secondary structure. It may be of phylogenetic interest that the sequence relationship of Dictyostelium porin to the two fungal porins is not closer than to the human porin. In fact, there are a number of identities that are unique to the Dictyostelium and human porins. The most conspicuous example is the sequence Ala-Lys-Val-Asn-Asn near amino acid 200.

Whereas the sequence differences between the Dictyoste- lium porin and other mitochondrial porins are considerable, functional similarities are striking. The most frequently ob- served single-channel conductance in 1 M KC1 was 4-4.5 nS. A second peak in the histogram comprised values of 2-2.5 nS (Fig. 3). Both single-channel conductances represent stable states of the channel. These states are typical of all mito- chondrial porins investigated, with the only exception of Paramecium porin which forms exclusively the 2.5-nS channel (17).

Our data suggest that Dictyostelium porin in its open state forms a wide water-filled channel and allow a rough estimate of the effective diameter of the pores. Assuming that the pores are filled with a solution of the same specific conductivity u

21078 Mitochondrial Porin from D. discoideum

scmmn UCEVPIN

HSPOPIN

DDPORIN

g: E T T

... "

"

"

P T

. . .

- W A - T L G V G A S P D T O K L D Q A - L p 8 # -

L T L " S A L L D G - K Y V Y A G

F V L G I U V Y T K U P K Q G Y T P

as the external solution, and assuming a cylindric pore with a length 1 of 6 nm, as suggested by electron microscopy (26, 27), the average pore diameter d can be calculated according to the following equation.

G = a d / 1 (2)

From the peak in the histogram shown in Fig. 3 ( G = 4 nS; a = 110 mS/cm in 1 mM KC1) an effective diameter (2r) of about 1.7 nm is obtained. This is close to the pore diameter of other mitochondrial porins, except of Paramecium porin (17). This value is also consistent with the results of x-ray and electron microscopic studies (26-28). 16 8-strands cross- ing the membrane, a common structure suggested for bacterial and mitochondrial porins (8,9), will also form a channel with an inner diameter of about 2 nm (8). This diameter allows the passage of hydrophilic solutes up to molecular masses of 2500-3000 Da. It has to be noted, however, that the assump- tion of 16 8-strands is still tentative, since recently a model for yeast porin has been proposed which contains only 12 8- strands (26,54).

The opening of the Dictyostelium porin channel proved to be voltage regulated; transmembrane potentials higher than 20 mV resulted in a reduced conductance. The steadv-state

polyanion of M, 10,000. This copolymer of methacrylate, maleate, and styrene at a ratio of 1:2:3 shifts rat liver porin toward the closed state which is impermeable for ADP, ATP, and creatine phosphate, but not for creatine (29, 30). Thus porin could regulate mitochondrial metabolism by controlling the permeability of the outer membrane in response to chang- ing transmembrane potentials. However, as yet no data are available that demonstrate a potential difference across the mitochondrial outer membrane. Because of the large pore size and the weak ion selectivity of porin channels, it is unlikely that such a potential exists (1). Nevertheless, it is possible that porin regulates permeability of the outer membrane in response to electric coupling between both membranes (29, 30). The potential of the inner mitochondrial membrane has been estimated to be 150 mV and, as a substrate for coupling, contact sites between both membranes have been observed in uiuo (30).

Acknowledgments-We thank Dr. J. L. M. Leunissen (Aurion, Wageningen, NL) for providing 1-nm gold conjugated anti-mouse antibodies prior to commercial release and Dr. F. P. Thinnes for providing part of Fig. 9. We gratefully acknowledge the expert tech- nical assistance of Iris Adrian, Monika Westphal, Barbara Fellerhoff, Barbara Rahn, Elke Biegelmann, and Ursula Saur.

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