Biochimica et Biophysica Acta, 1039 (1990) 73-80
73
Elsevier BBAPRO 33651
Isolation of rat liver spectrin and identification of functional domains
1,
2,
Rocco Falchetto S a b i n a L e u t e n e g g e r 1, O r i o l B a c h s J o a n S e r r a t o s a 2, Yvonne Bloemhard a and Paolo Gazzotti 1 Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich (Switzerland) and 2 Department of Cell Biology, University of Barcelona, Barcelona (Spain)
(Received 1 September1989) (Revised manuscript received14 February 1990)
Key words: Spectrin; Cytoskeleton;Actin binding; Calmodulin binding; (Rat liver)
Immunohistochemical studies carried out with liver sections show that spectrin is uniformly distributed along the whole plasma membrane of hepatocytes. The bilecanalicular spectrin is released during the purification of liver subplasma membrane fractions, whereas most of the basolateral spectrin remains tightly bound to the membrane. Spectrin associated with the basolateral membranes has been purified and its subunits isolated. The a-subunit retains the ability to bind both calmodulin and actin. Fragments have been obtained either by chemical or by proteolytical digestion of the 240 kDa a-subunit. Treatment with CNBr yields fragments of about 30 kDa which bind actin and calmodulin. Digestion with Staphylococcus aureus V-8 proteinase yields a calmodulin-binding fragment of 27 kDa and an actin-binding fragment of 31 kDa.
Introduction Spectrins are a family of structurally and functionally related proteins which represent the major component of the cytoskeletal network underlying the plasma membrane [1]. These proteins are rod-like molecules made of two non-identical subunits. The subunits of erythrocyte spectrin have molecular masses of 240 (asubunit) and 220 kDa (fl-subunit), whereas those of brain spectrin (also called fodrin) are 240 and 235 kDa. Spectrin binds to the membrane mainly in the form of tetramers linked together by actin oligomers. A common structural feature of spectrins is a repeat sequence of 106 amino acids which makes up most of the molecule [2]. The erythroid and nonerythroid ~t-subunits present structural dissimilarity, especially in the carboxy-terminal domain [3], and they share only limited immunological cross-reactivity [4]. In addition, the a-
Abbreviations: PMSF, phenylmethylsulfonylfluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel eleetrophoresis; EDTA, ethylenediaminetetraaceticacid; PBS, 140 mM NaCl, 10 mM sodium phosphate (pH 7.0). Correspondence: P. Gazzotti, Laboratory of Biochemsitry, ETHZentrum CH-8092 Zurich, Switzerland.
subunit of the nonerythroid spectrin has the ability to bind calmodulin [5]. The location of the calmodulinbinding domain in the a-subunit of brain spectrin is still a matter of debate. Rotary shadowing electron microscopy studies show a binding site for calmodulin close to the amino terminus of the a-subunit [6]. On the other hand, peptide mapping studies [7] and primary structure determination [8] of brain spectrin place the calmodulin-binding site in the mid-region of the molecule. Work carried out using cloning and deletion mutagenesis and recombinant peptides [9,10] seems to confirm this location. Finally, analysis of the primary structure of the a-subunit of brain spectrin predicts a typical calmodulin-binding site at the carboxy-terminus of the molecule [11]. Until now, most of the research on spectrin has been carried out with brain or erythrocytes. We have decided to study liver spectrin in order to gain an understanding of its structure and its functional role in this tissue. It has been previously reported that liver spectrin has the same subunit molecular mass as that of brain [12]. In the first part of this work we have investigated the distribution of liver spectrin in the different functional domains of the plasma membrane. Spectrin has then been purified and its two subunits separated. Chemical and proteolytical fragmentation of the a-subunit has been carried out in an attempt to identify the calmodulin- and actin-binding regions.
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
74 Materials and Methods
Materials Staphylococcus aureus V-8 proteinase was from Boehringer-Mannheim. Cyanogen bromide was from Fluka (puriss.). Calmodulin was purified from bovine brains [13]. Actin was purified from rat liver [12]. Enzymobeads for 125I-labelling of proteins were from BioRad Laboratories.
Membrane preparation Young female rats fasted 20 h were used in this study. The livers were perfused with 154 mM NaC1 and homogenized with five up-and-down strokes of a Dounce homogenizer, fitted with a loose pestle. Plasma membranes were prepared from the homogenate using percoll gradients as previously described [12]. Subplasmamembrane fractions enriched in the different functional domains of liver plasma membrane were obtained by homogenizing the suspension of plasma membranes with sixty up-and-down strokes of a Dounce homogenizer fitted with a tight pestle. The membranes were then separated by centrifugation on a discontinuous sucrose gradient [12]. Three fractions were collected: BC (interface 34.5% w / v , contains mainly the canalicular domain); BL (interface 39.5-44.5%, basolateral domain); and HBL (pellet, contains heavy basolateral membranes enriched in junctional proteins).
Isolation of lioer spectrin Liver spectrin was isolated from either the whole plasma membranes or from the heavy basolateral fraction. The membranes, at a concentration of 15 mg p r o t e i n / m l , were incubated 10 min on ice with 3 vol. of solubilization buffer containing 150 mM KC1, 5 mM Tris-HCl, 2 mM MgCI2, 1 mM ATP, 5 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.33% Triton X-100 (final p H 7.5). After centrifugation at 100000 x g for 40 min the pellet was resuspended in alkaline buffer containing 800 mM NaC1, 1 mM EDTA, 10% glycerol, 5 mM sodium pyrophosphate, 0.1 mM PMSF and 0.5 mM D T T (final pH 8.0) at a final volume of 1.5 ml per 20 mg of original plasma membranes. The pH of the suspension was then adjusted to 9.0 with 1 M NaOH. After 30 min incubation on ice, the suspension was centrifuged at 100000 x g for 30 min. The alkaline supernatant, containing about 50% of the plasma membrane spectrin, was collected and 15 mM sodium pyrophosphate (pH 8.0) was added. Spectrin in the alkaline supernatant was further purified by gel filtration in a Sepharose CL-4B column equilibrated with buffer 1 containing 800 mM NaC1, 2 mM K-EDTA, 10 mM sodium pyrophosphate, 0.1 mM PMSF and 0.5 mM D T T (final pH 8.0). Proteins were eluted from the column with the same buffer, and the spectrin content of the eluted fractions tested by SDS-
polyacrylamide gel electrophoresis. The fractions enriched in spectrin were pooled and precipitated by the addition of 250 mg ammonium sulfate per ml of suspension under gentle stirring. After 20 min incubation on ice the suspension was centrifuged at 40 000 x g for 20 min. The pellet was resuspended in a small volume of second gel filtration buffer: 1 M NaBr, 2 mM K-EDTA, 1 mM NAN3, 15 mM sodium pyrophosphate, 10 mM sodium phosphate, 0.5 mM D T T and 0.1 mM PMSF (final pH 8.2). This sample was applied to a Sepharose CL-4B column equilibrated with the second gel filtration buffer. After elution, the spectrin enriched fractions were pooled and precipitated with 200 m g / m l ammonium sulfate as described above. The pellet was resuspended in 10 mM Tris-HCl, 3 mM NaN 3, 0.1 mM DTT, 0.1 mM CaC12 (pH 8.2) and dialyzed overnight at 4 ° C against 100 volumes of the same buffer. The turbidity formed during dialysis was eliminated by centrifugation of the suspension at 100000 x g for 2 h. The pure spectrin in the supernatant was concentrated by dialysis against a 30% solution of poly(ethylene glycol) 20 000 and stored at - 80 ° C.
Isolation of the a-subunit The a-subunit of spectrin was isolated by calmodulin affinity chromatography after dissociation of the two subunits with 2 M NaI as previously described [14]. In some experiments a crude fraction of liver spectrin was used to isolate the ct-subunit by preparative gel electrophoresis and electroelution. The proteins were suspended in electrophoresis sample buffer (1 mg protein per ml) and 2.5 ml of this suspension were loaded on a 1.5 mm thick 4% SDS-polyacrylamide preparative gel. The proteins were separated by overnight electrophoresis at 30 V. The a-subunit was visualized using Coomassie brilliant blue and cut out. Electroelution was carried out in a Bio-Rad Electro-Eluter (3.5 h at 8 m A / t u b e ) using a buffer containing: 25 mM Tris-HC1, 192 mM glycine, 0.1% SDS and 1 mM DTT. After electroelution SDS was removed from the sample as previously described [15].
Polyacrylamide gel electrophoresis SDS-polyacrylamide electrophoresis was normally carried out according to Laemmli [16]. The electrophoretic separation and analysis of the digested proteins was carded out using Tricine buffer gels [17].
Protein electroblotting. The electrophoretic transfer of proteins from the polyacrylamide gel to nitrocellulose sheets was carried out as previously described [18] using a transfer buffer containing: 192 mM glycine, 25 m M Tris-HC1, 0.02% SDS and 20% methanol. Gels containing high molecular mass proteins were blotted overnight at 30 V (constant). The efficiency of the electrotransfer was checked by
75 staining part of the nitrocellulose with Amido black. The proteins remaining in the gel after transfer were stained with Coomassie brilliant blue. These controls are important since the electrotransfer of spectrin is not easy and only a small part of the spectrin doublet is normally transferred to the nitrocellulose. Furthermore, the conditions used for SDS-PAGE might also determine the extent of transfer. In fact, we have observed that in the absence of D T T or mercaptoethanol in the sample buffer only the fl-chain is electrotransferred.
Radioiodination of calmodulin and actin Liver actin and brain calmodulin were enzymatically iodinated using the Enzymobead reagent from Bio-Rad. Radioiodination of calmodulin was carried out in 50 mM sodium phosphate (pH 7.0) and 100 /~M CaCI 2. Liver actin was iodinated in the same buffer containing 1 mM MgC12 instead of CaC12 (azide present in the actin storage buffer was removed by dialysis before iodination). The specific radioactivity of the radioiodinated actin was always rather low. The amount of radioactivity incorporated could be increased using the Chloramine T method. However, the protein denaturates and it is no longer suitable for our studies. Calmodulin, actin and antibodies ooerlay The nitrocellulose sheets containing the blotted proteins were incubated at room temperature with 2% defatted milk powder in PBS (140 mM NaC1, 10 mM sodium phosphate (pH 7.0)) for 2 h. The sheets were then washed twice with 0.1% defatted milk powder in PBS. [125I]Calmodulin overlay was carried out at room temperature by incubating the sheets in a solution of 0.1% defatted milk powder containing 300 /LM CaC12 and 5 nM radioactive calmodulin (5 nCi/pmol). The sheets were then washed three times with the same solution without calmodulin and after air-drying autoradiography was carried out using X-ray Kodak films. Overlay with [125I]actin was carried out as described above, but no CaC12 was added to the solution. For the visualization of the antibody reactions, the nitrocellulose sheets were blocked as described above and subsequently incubated 1 h with 2% milk p o w d e r / P B S containing the anti-spectrin antibodies in a 1 : 200 dilution. After washing with same solution without the antibodies, the sheets were incubated with a peroxidase-labelled second antibody (1 : 800) for 1 h. The reaction was then visualized with 4-chloro-l-naphtol. CNBr digestion of the a-subunit of lioer spectrin The t~-subunit of spectrin was fragmented with CNBr essentially as previously described [19]. The protein purified by calmodulin-affinity chromatography was precipitated with 8% TCA (in the presence of deoxycholate) at 4 ° C , washed once with acetone/HC1 and once with cyclohexane. The precipitate was solublized
by sonication in 70% formic acid, and digested with CNBr, under nitrogen, for 24 h at room temperature in the dark. The formic acid was evaporated at 4 5 ° C under nitrogen and the digest was washed twice with 300/~1 of water and finally suspended in electrophoresis sample buffer. The ~t-subunit obtained by electroelution was treated in the same way except that after removal of SDS the protein was directly solubilized in 70% formic acid.
Proteolysis in situ Proteolytic fragmentation of the a-subunit of spect i n has been carried out in situ using a procedure slightly modified from previously published methods [20]. a-Subunit-containing gel slices obtained by preparative gel electrophoresis were loaded on tricine buffer gels and overlayed with 70 /zl 'slice buffer' (50 mM Tris-HC1, 20% glycerol, 0.1% SDS (final p H 6.8)) containing different concentrations of S. aureus V-8 proteinase. After 15 min incubation, electrophoresis (at 70 V) was carried out until the sample reached the stacking gel/running gel interface and was then stopped for 15 min to allow protein digestion. Electrophoresis was then continued again overnight at 30 V. Production of anti-spectrin antibodies In this study the antigen was an a-spectrin which was freed from contaminants using preparative SDS-PAGE. The band was then cut out and the protein electroeluted. The purified protein (0.4 mg) was emulsified in 0.4 ml complete Freund's adjuvant, and intramuscularly injected into the back of several rabbits. A second injection was given 10 days later. Subcutaneous booster injections (0.4 mg a-spectrin in incomplete Freund's adjuvant) were given 6 weeks later. 10 days after the booster injections the rabbits were bled. More blood was then withdrawn at three day intervals. Immunocytochemical oisualization of a-spectrin Pieces of liver were rapidly frozen in liquid nitrogen and 4 /~m sections were cut using a cryotom. The sections were incubated for 30 min in PBS containing normal porcine serum (1:10), new born calf serum (1:10) and 0.01% sodium azide. After draining, the sections were incubated at room temperature for 1 h with the anti-a-spectrin antibodies diluted 1 : 40 in PBS containing new born calf serum (1:10) and 0.01% sodium. After washing with PBS (three times 10 min) the second antibody (porcine anti-rabbit diluted 1 : 80) was applied for 1 h. Finally, the sections were incubated for 1 h with rabbit peroxidase anti-peroxidase (PAP) diluted 1:80. To visualize the antibody reaction, the sections were incubated with diaminobenzidine (25 m g / m l in PBS (pH 7.6) containing 0.03% hydrogen peroxide).
76
Other methods Protein determination was carried out using a modified Lowry procedure [21]. Results
Localization of spectrin in the different functional domains of liver plasma membranes Spectrin present in rat liver subplasmamembrane fractions enriched in the different functional domains has been identified either by looking at the presence of the high molecular mass doublet after separation of the proteins by SDS-polyacrylamide gel electrophoresis or by [t25I]calmodulin overlay of the blotted proteins to visualize the a-subunit (Fig. 1, A and B). The results show that spectrin is mainly associated with the basolateral and the heavy basolateral fractions, and very little is present in the canalicular membrane fraction. A possible explanation for the lack of spectrin in the canalicular membrane is that this protein dissociates during the preparation of the membrane fractions. Indeed, we have observed that the membrane skeleton can easily dissociate from the plasma membrane depending on the medium used during the preparation. The spectrin which dissociates from the plasma membrane during homogenization can be recovered in the soluble fraction obtained after sedimenting the microsomes. For instance, the hypotonic bicarbonate-medium which is commonly used for the isolation of liver plasma membranes gives membranes which are almost devoid of spectrin (data not shown). On the other hand, the magnesium-containing isotonic medium that we have used in these experiments seems to be particularly suita-
ble in preventing dissociation of the membrane cytoskeleton. To further investigate the distribution of spectrin in the plasma membrane we have prepared antibodies against the a-subunit of liver spectrin and carried out an immunohistochemistry study. Immunoblotting experiments using either the whole plasma membrane or the isolated a-subunit clearly show that the antibodies reacted specifically with the a-subunit (Fig. 2). The immunohistochemical results shown in Fig. 3A seem to indicate that the antibodies react with the whole plasma membrane, including the canalicular region. No staining was observed in the control incubated with the preimmune serum from the same rabbit (Fig. 3B). Additional immunohistochemical studies carried out with affinity purified antibodies confirmed these results. The anti-aspectrin antibodies seem to react also with the nuclear envelope confirming a previously published observation which showed the presence of spectrin-like proteins in the nucleus [22].
Isolation of rat liver spectrin and separation of the two subunits We have shown that liver plasma membranes prepared in isotonic medium still retain most of their cytoskeleton. Five major components of this membrane associated cytoskeleton have been previously identified [23]. The first two are two polypeptides (52 and 56 kDa) which compose the intermediate filaments associated with desmosomes, the third is actin (43 kDa) and the last two are the two high molecular mass polypeptides which constitute spectrin (240 and 235 kDa). These proteins can also be recognized in the gel electrophoresis protein pattern of our liver plasma membrane (Fig.
A
B
kDa 200 116 97 66
45
0
1
2
3
4
1
2
3
4
Fig. 1. SDS/(4-12q$)-PAGE and [1251]calmodulin overlay of subplasmamembrane fractions. (A) SDS-PAGE pattern of membrane proteins (70 #g). Lane 1, whole plasma membrane; lane 2, bile canalicular membranes; lane 3, basolateral membranes; lane 4, heavy basolateral membranes; and lane 0, molecular mass standards from Bio-Rad. (B) Autoradiogram of the gel in A after [125I]calmodulin overlay.
77
kDa 240
were separated by gel permeation chromatography at high ionic strength and the fractions containing the partially purified spectrin were collected (Fig. 4, lane 6). The contaminants still associated with spectrin could be finally removed by centrifugation following dialysis against low ionic strength buffer. The two subunits of spectrin were dissociated by partial denaturation at a high concentration of sodium iodine, and the c~-subunit separated by means of calmodulin-affinity chromatography (Fig. 4, lanes 8 and 9). As shown in Fig. 5, the isolated a-subunit still maintains its ability to bind calmodulin and actin. The binding of [12SI]calmodulin is calcium-dependent and was not prevented by preincubating with a large amount of cold actin. Similarly, [125I]actin binding was not prevented by preincubating with cold calmodulin (data not shown) indicating that the two proteins do not bind to the same site.
~--
1
2
Fig. 2. Immunoblotting using anti-a-spectrin antibodies. Lane 1, whole plasma membranes; and lane 2, purified a-subunit of spectrin. The experimentalconditionsare describedin 'Materials and Methods'.
4, lane 1). In order to purify liver spectrin the basolateral membranes were first solubilized with Triton X-100 to remove most of the integral proteins. Actin and about 50% of spectrin could then be solubilized from Triton X-100 insoluble pellet by extraction with a high ionic strength alkaline medium (Fig. 4, lane 5). Most of the intermediate filaments remained in the pellet. Actin and spectrin present in the alkaline extract
Chemical and proteolytical fragmentation of the a-subunit of liver spectrin The location of the calmodulin- and actin-binding domains in the a-subunit of spectrin has been investigated by specific cleavage of the molecule either by chemical methods or by the use of proteinases. Cleavage at methionine residues with CNBr, at low concentration, gives a major calmodulin-binding fragment of 150 kDa (data not shown). At higher CNBr concentration a large number of fragments with a molecular mass below 66 kDa are generated (Fig. 6A). Several of these fragments still bind calmodulin, however, particularly a strong reaction was observed with fragments of 34, 32
~iii ~i
~i!i~i~i~iii!iiiii~i !iii ~i!iiiiiii;¸~i;i
Fig. 3. Immunohistochemistryof rat liver sections. The sections were prepared as described in 'Materials and Methods' and stained with either anti-a-spectrin antibodies (A) or preimmune serum (B). The scale bar corresponds to 10/xm.
78
iiiiiiiiiiiiii!iiiiiiiil;~iil
mm
0
1
2
34
5
6
7
89
Fig. 4. SDS/(4-I2~)-PAGE of the different fractions (70/~g) obtained during the purification of liver spectrin and the isolation of the a-subunit. Lane 1, whole plasmamembrane; lane 2, Triton X-100 supernatant; lane 3, Triton X-100 pellet; lane 4, alkaline pellet; lane 5, alkaline extract; lane 6, spectrin after the two gel filtration steps; lane 7, purified spectrin; lane 8, spectrin a-subunit; lane 9, spectrin fl-subunit; and lane 0, molecular mass standards.
and 23 kDa (Fig. 6B). At lower amounts of CNBr the 34 and 32 kDa fragments are better resolved (Fig. 7A), but no 23 kDa fragment is generated. Fig. 7 seems to indicate that the fragments which bind calmodulin can also bind actin. However, the presence of different fragments migrating in the same position can not be excluded. Calmodulin- and actin-binding fragments
A
B
having different molecular masses can be obtained by proteolysis of the a-spectrin with S. aureus V-8 proteinase. As shown in Fig. 8A, lane 1, if the digestion is carried out with a low amount of proteinase several calmodulin-binding fragments are generated, the smallest having a molecular mass of about 60 kDa. A 10-fold increase in the amount of proteinase causes the disappearance of many of the high molecular mass calmodulin-binding fragments, and the formation of a new calmodulin-binding fragment of 27 kDa (Fig. 8A, lane 2) and of an actin-binding fragment of 31 kDa (Fig. 8B, lane 2, indicated by the arrow). Discussion
Fig. 5. Autoradiograms of [12SI]calmodulin (A) and [125I]actin (B) overlays of the a-subunit of liver spectrin isolated by calmodulin-affinity chromatography.
Liver plasma membrane is a highly specialized membrane which contains three different functional domains. The membrane cytoskeleton which has been found associated with these domains has different functional and structural properties [24]. The immunocytochemical studies reported here show that spectrin is a common component of the membrane skeleton since it is uniformly distributed along the whole plasma membrane of hepatocytes. However, the way spectrin interacts with the membrane skeleton in the functional domains might be different. In fact, we have observed that sp.ectrin associated with the canalicular domain is easily released during the isolation of the subplasmamembrane fractions, whereas most of the basolateral
79
A
B
A
B
9~
61 4,' 3; .
1 Fig. 6. SDS-PAGE and calmodulin overlay of CNBr fragments of the a-subunit. (A) Right lane, Coomassie blue staining of the fragments obtained by CNBr digestion of the a-subunit using 3 #g CNBr per #g protein. The fragments were separated on 16.5% Tricine gels. Left lane, molecular mass standards. (B) Autoradiogram of the [12sI]calmodulin overlay carried out with the same CNBr fragments used in A. The left lane shows 14C-labelled low molecular mass standards (92, 55, 46, 30 and 14 kDa).
A
B
k Da 92-. 46-30-.
14,,-
¸I¸
i ¸¸
Fig. 7. Calmodulin and actin overlay of CNBr fragments obtained from the a-subunit of spectrin. Digestion was carried out at 1.6 pg CNBr per #g protein. (A) [12Sl]calmodulin overlay. (B) [l:Sl]actin overlay.
2
1
2
Fig. 8. 'In situ' proteolysis of the a-subunit of spectrin with S. a u r e u s V-8 proteinase. Gel slices containing about 40 pg of a-subunit were loaded on the gel and digested with either 70 ng V-8 proteinase (lane 1) or 700 ng V-8 proteinase (lane 2). (A) [125I]calmodulinoverlay. (B) [125I]actin overlay.
spectrin remains tightly b o u n d to the membrane. The basolateral spectrin can be extracted f r o m the membrane by alkaline treatment at high ionic strength. Using this procedure only half of the b o u n d spectrin is released, indicating that also in this m e m b r a n e different types of spectrin association might occur. A n interesting possibility which we are currently investigating is the existence of different isoforms of spectrin associated with the plasma m e m b r a n e of hepatocytes. Spectrin isoforms have already been f o u n d in other epithelial tissues and in brain [25,26]. Liver spectrin has been purified from the alkaline extract of basolateral m e m branes. The major c o n t a m i n a n t s f o u n d associated with spectrin are actin and a 200 k D a protein. The 200 k D a protein is tightly associated with spectrin and can only be removed in the last purification step since it gives insoluble aggregates when dialyzed against a low ionic strength buffer. Most likely this protein is analogous to red blood cell ankyrin, a protein involved in the binding of spectrin to the membrane. The a-subunit of liver spectrin still retains its ability to bind b o t h actin and calmodulin and can therefore be easily purified by calmodulin-affinity c h r o m a t o g r a p h y after partial denaturation of spectrin to allow dissociation of the two subunits. The a-subunit plays an i m p o r t a n t role in the regulation of spectrin function since it contains the binding site for calmodulin and actin. The localization of the calmodulin-binding site in the a-subunit is still a matter of debate, since different calmodulin-binding regions have been p r o p o s e d [6-10]. These different find-
80 ings could be explained by the presence of more than one binding site for calmodulin as it has been shown with caldesmon, another cytoskeletal calmodulin and actin-binding protein which binds two molecules of calmodulin [27]. However, the available studies indicate that the ct-subunit of brain can only bind one molecule [28]. Our studies on the identification of the calmodulin- and actin-binding site of the a-subunit were mainly directed towards the isolation of small calmodulin- and actin-binding fragments which could then be sequenced. The digestion of the a-subunit with high concentrations of S. aureus V-8 proteinase separates the two binding domains generating a 31 kDa fragfiaent which has lost the ability to bind calmodulin anda 27 kDa fragment which binds exclusively calmodulin. On the other hand, the digestion of the a-subunit with CNBr gives several fragments with a molecular mass of around 30 kDa which bind calmodulin a n d / o r actin. Whether the calmodulin and actin domains are localized on the same CNBr fragments can not be decided on the basis of our experiments. To get a conclusive answer a more detailed investigation using two-dimensional gel electrophoresis is needed.
Acknowledgements We wish to thank Prof. E. Carafoli for encouragement and helpful discussions. This work was supported by the Swiss National fond (Grant No. 3.531.-0.86).
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6 Tsukita, S., Tsukita, S., Ishikawa, H., Kurokawa, M., Morimoto, K., Sobue, K. and Kakiuchi, S. (1983) J. Cell Biol. 97, 574-578. 7 Harris, A.S. and Morrow, J.S. (1988) J. Neurosci. 8, 2640-2651. 8 Harris, A.S., Croall, D.E. and Morrow, J.S. (1988) J. Biol. Chem. 263, 15754-15761. 9 Leto, T.L., Pleasic, S., Forget, B.G., Benz, E.J., Jr. and Marchesi, V.T. (1989) J. Biol. Chem. 264, 5826-5830. 10 Sri Widada, J., Asselin, J., Colote, S., Marti, J., Ferraz, C., Trave, G., Heiech, J. and Liautard, J.-P. (1989) J. Mol. Biol. 205, 455-458. 11 Wasenius, V.M., Saraste, M., Salven, P., Er~imaa, M., Holm, L. and Letho, V.P. (1989) J. Cell. Biol. 108, 79-93. 12 Amrein-Oloor, M. and Gazzotti, P. (1987) Biochem. Biophys. Res. Commun. 145, 1033-1037. 13 Gopalakrishna, R. and Anderson, W.B. (1982) Biochem. Biophys. Res. Commun. 104, 830-836. 14 Glenney, J.R., Jr. and Weber, K. (1985) Anal. Biochem. 150, 364-368. 15 Konisberg, W.H. and Henderson, L. (1983) Methods Enzymol. 91, 254-259. 16 Laemmli, U.K. (1970) Nature 227, 680-685. 17 Schagger, H. and Von Jagow, G. (1987) Anal. Biochem. 166, 368-379. 18 Towbin, H., St~laelin, T. and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA 76, 4350-4354. 19 James, P., Maeda, M., Fischer, R., Verma, A.K., Krebs, J., Penniston, J.T. and Carafoli, E. (1988) J. Biol. Chem. 2905-2910. 20 Cleveland, D.W., Fischer, S.G., Kirschner, M.W. and Laemmli, U.K. (1977) J. Biol. Chem. 252, 1102-1106. 21 Markwell, M.K., Haas, S., Tolbert, N.E. and Bieber, L.L. (1981) Methods Enzymol. 72, 296-303. 22 Bachs, O. and Carafoli, E. (1987) J. Biol. Chem. 262, 10786-10790. 23 Hubbard, A.L. and Ma, A. (1983) J. Cell Biol. 96, 230-239. 24 Phillips, M.J. and Satir, P. (1988) in The Liver, Biology and Pathology (Arias, I.M., Jakoby, W.B., Popper, H., Schachter, D. and Shafritz, D.A., eds.), pp. 11-29, Raven Press, New York. 25 Virtanen, I., Thornell, L.-E., Damjanov, I., Hormia, M. and Letho, V.-P. (1986) J. Cell. Biochem. 30, 339-356. 26 Vanderpuye, O.A., Kelley, L.K., Morrison, M.M. and Smith, C.H. (1988) Biochim. Biophys. Acta 943, 277-287. 27 Wang, C.L.A., Wang, L.W.C. and Lu, R.C. (1989) Biochem. Biophys. Res. Commun. 162, 746-752. 28 Harris, A.S., Anderson, J.P., Yurchenco, P.D., Green, L.A.D., Ainger, K.J. and Morrow, J.S. (1986) J. Cell. Biochem. 30, 51-69.
73
Elsevier BBAPRO 33651
Isolation of rat liver spectrin and identification of functional domains
1,
2,
Rocco Falchetto S a b i n a L e u t e n e g g e r 1, O r i o l B a c h s J o a n S e r r a t o s a 2, Yvonne Bloemhard a and Paolo Gazzotti 1 Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich (Switzerland) and 2 Department of Cell Biology, University of Barcelona, Barcelona (Spain)
(Received 1 September1989) (Revised manuscript received14 February 1990)
Key words: Spectrin; Cytoskeleton;Actin binding; Calmodulin binding; (Rat liver)
Immunohistochemical studies carried out with liver sections show that spectrin is uniformly distributed along the whole plasma membrane of hepatocytes. The bilecanalicular spectrin is released during the purification of liver subplasma membrane fractions, whereas most of the basolateral spectrin remains tightly bound to the membrane. Spectrin associated with the basolateral membranes has been purified and its subunits isolated. The a-subunit retains the ability to bind both calmodulin and actin. Fragments have been obtained either by chemical or by proteolytical digestion of the 240 kDa a-subunit. Treatment with CNBr yields fragments of about 30 kDa which bind actin and calmodulin. Digestion with Staphylococcus aureus V-8 proteinase yields a calmodulin-binding fragment of 27 kDa and an actin-binding fragment of 31 kDa.
Introduction Spectrins are a family of structurally and functionally related proteins which represent the major component of the cytoskeletal network underlying the plasma membrane [1]. These proteins are rod-like molecules made of two non-identical subunits. The subunits of erythrocyte spectrin have molecular masses of 240 (asubunit) and 220 kDa (fl-subunit), whereas those of brain spectrin (also called fodrin) are 240 and 235 kDa. Spectrin binds to the membrane mainly in the form of tetramers linked together by actin oligomers. A common structural feature of spectrins is a repeat sequence of 106 amino acids which makes up most of the molecule [2]. The erythroid and nonerythroid ~t-subunits present structural dissimilarity, especially in the carboxy-terminal domain [3], and they share only limited immunological cross-reactivity [4]. In addition, the a-
Abbreviations: PMSF, phenylmethylsulfonylfluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel eleetrophoresis; EDTA, ethylenediaminetetraaceticacid; PBS, 140 mM NaCl, 10 mM sodium phosphate (pH 7.0). Correspondence: P. Gazzotti, Laboratory of Biochemsitry, ETHZentrum CH-8092 Zurich, Switzerland.
subunit of the nonerythroid spectrin has the ability to bind calmodulin [5]. The location of the calmodulinbinding domain in the a-subunit of brain spectrin is still a matter of debate. Rotary shadowing electron microscopy studies show a binding site for calmodulin close to the amino terminus of the a-subunit [6]. On the other hand, peptide mapping studies [7] and primary structure determination [8] of brain spectrin place the calmodulin-binding site in the mid-region of the molecule. Work carried out using cloning and deletion mutagenesis and recombinant peptides [9,10] seems to confirm this location. Finally, analysis of the primary structure of the a-subunit of brain spectrin predicts a typical calmodulin-binding site at the carboxy-terminus of the molecule [11]. Until now, most of the research on spectrin has been carried out with brain or erythrocytes. We have decided to study liver spectrin in order to gain an understanding of its structure and its functional role in this tissue. It has been previously reported that liver spectrin has the same subunit molecular mass as that of brain [12]. In the first part of this work we have investigated the distribution of liver spectrin in the different functional domains of the plasma membrane. Spectrin has then been purified and its two subunits separated. Chemical and proteolytical fragmentation of the a-subunit has been carried out in an attempt to identify the calmodulin- and actin-binding regions.
0167-4838/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
74 Materials and Methods
Materials Staphylococcus aureus V-8 proteinase was from Boehringer-Mannheim. Cyanogen bromide was from Fluka (puriss.). Calmodulin was purified from bovine brains [13]. Actin was purified from rat liver [12]. Enzymobeads for 125I-labelling of proteins were from BioRad Laboratories.
Membrane preparation Young female rats fasted 20 h were used in this study. The livers were perfused with 154 mM NaC1 and homogenized with five up-and-down strokes of a Dounce homogenizer, fitted with a loose pestle. Plasma membranes were prepared from the homogenate using percoll gradients as previously described [12]. Subplasmamembrane fractions enriched in the different functional domains of liver plasma membrane were obtained by homogenizing the suspension of plasma membranes with sixty up-and-down strokes of a Dounce homogenizer fitted with a tight pestle. The membranes were then separated by centrifugation on a discontinuous sucrose gradient [12]. Three fractions were collected: BC (interface 34.5% w / v , contains mainly the canalicular domain); BL (interface 39.5-44.5%, basolateral domain); and HBL (pellet, contains heavy basolateral membranes enriched in junctional proteins).
Isolation of lioer spectrin Liver spectrin was isolated from either the whole plasma membranes or from the heavy basolateral fraction. The membranes, at a concentration of 15 mg p r o t e i n / m l , were incubated 10 min on ice with 3 vol. of solubilization buffer containing 150 mM KC1, 5 mM Tris-HCl, 2 mM MgCI2, 1 mM ATP, 5 mM sodium pyrophosphate, 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 0.33% Triton X-100 (final p H 7.5). After centrifugation at 100000 x g for 40 min the pellet was resuspended in alkaline buffer containing 800 mM NaC1, 1 mM EDTA, 10% glycerol, 5 mM sodium pyrophosphate, 0.1 mM PMSF and 0.5 mM D T T (final pH 8.0) at a final volume of 1.5 ml per 20 mg of original plasma membranes. The pH of the suspension was then adjusted to 9.0 with 1 M NaOH. After 30 min incubation on ice, the suspension was centrifuged at 100000 x g for 30 min. The alkaline supernatant, containing about 50% of the plasma membrane spectrin, was collected and 15 mM sodium pyrophosphate (pH 8.0) was added. Spectrin in the alkaline supernatant was further purified by gel filtration in a Sepharose CL-4B column equilibrated with buffer 1 containing 800 mM NaC1, 2 mM K-EDTA, 10 mM sodium pyrophosphate, 0.1 mM PMSF and 0.5 mM D T T (final pH 8.0). Proteins were eluted from the column with the same buffer, and the spectrin content of the eluted fractions tested by SDS-
polyacrylamide gel electrophoresis. The fractions enriched in spectrin were pooled and precipitated by the addition of 250 mg ammonium sulfate per ml of suspension under gentle stirring. After 20 min incubation on ice the suspension was centrifuged at 40 000 x g for 20 min. The pellet was resuspended in a small volume of second gel filtration buffer: 1 M NaBr, 2 mM K-EDTA, 1 mM NAN3, 15 mM sodium pyrophosphate, 10 mM sodium phosphate, 0.5 mM D T T and 0.1 mM PMSF (final pH 8.2). This sample was applied to a Sepharose CL-4B column equilibrated with the second gel filtration buffer. After elution, the spectrin enriched fractions were pooled and precipitated with 200 m g / m l ammonium sulfate as described above. The pellet was resuspended in 10 mM Tris-HCl, 3 mM NaN 3, 0.1 mM DTT, 0.1 mM CaC12 (pH 8.2) and dialyzed overnight at 4 ° C against 100 volumes of the same buffer. The turbidity formed during dialysis was eliminated by centrifugation of the suspension at 100000 x g for 2 h. The pure spectrin in the supernatant was concentrated by dialysis against a 30% solution of poly(ethylene glycol) 20 000 and stored at - 80 ° C.
Isolation of the a-subunit The a-subunit of spectrin was isolated by calmodulin affinity chromatography after dissociation of the two subunits with 2 M NaI as previously described [14]. In some experiments a crude fraction of liver spectrin was used to isolate the ct-subunit by preparative gel electrophoresis and electroelution. The proteins were suspended in electrophoresis sample buffer (1 mg protein per ml) and 2.5 ml of this suspension were loaded on a 1.5 mm thick 4% SDS-polyacrylamide preparative gel. The proteins were separated by overnight electrophoresis at 30 V. The a-subunit was visualized using Coomassie brilliant blue and cut out. Electroelution was carried out in a Bio-Rad Electro-Eluter (3.5 h at 8 m A / t u b e ) using a buffer containing: 25 mM Tris-HC1, 192 mM glycine, 0.1% SDS and 1 mM DTT. After electroelution SDS was removed from the sample as previously described [15].
Polyacrylamide gel electrophoresis SDS-polyacrylamide electrophoresis was normally carried out according to Laemmli [16]. The electrophoretic separation and analysis of the digested proteins was carded out using Tricine buffer gels [17].
Protein electroblotting. The electrophoretic transfer of proteins from the polyacrylamide gel to nitrocellulose sheets was carried out as previously described [18] using a transfer buffer containing: 192 mM glycine, 25 m M Tris-HC1, 0.02% SDS and 20% methanol. Gels containing high molecular mass proteins were blotted overnight at 30 V (constant). The efficiency of the electrotransfer was checked by
75 staining part of the nitrocellulose with Amido black. The proteins remaining in the gel after transfer were stained with Coomassie brilliant blue. These controls are important since the electrotransfer of spectrin is not easy and only a small part of the spectrin doublet is normally transferred to the nitrocellulose. Furthermore, the conditions used for SDS-PAGE might also determine the extent of transfer. In fact, we have observed that in the absence of D T T or mercaptoethanol in the sample buffer only the fl-chain is electrotransferred.
Radioiodination of calmodulin and actin Liver actin and brain calmodulin were enzymatically iodinated using the Enzymobead reagent from Bio-Rad. Radioiodination of calmodulin was carried out in 50 mM sodium phosphate (pH 7.0) and 100 /~M CaCI 2. Liver actin was iodinated in the same buffer containing 1 mM MgC12 instead of CaC12 (azide present in the actin storage buffer was removed by dialysis before iodination). The specific radioactivity of the radioiodinated actin was always rather low. The amount of radioactivity incorporated could be increased using the Chloramine T method. However, the protein denaturates and it is no longer suitable for our studies. Calmodulin, actin and antibodies ooerlay The nitrocellulose sheets containing the blotted proteins were incubated at room temperature with 2% defatted milk powder in PBS (140 mM NaC1, 10 mM sodium phosphate (pH 7.0)) for 2 h. The sheets were then washed twice with 0.1% defatted milk powder in PBS. [125I]Calmodulin overlay was carried out at room temperature by incubating the sheets in a solution of 0.1% defatted milk powder containing 300 /LM CaC12 and 5 nM radioactive calmodulin (5 nCi/pmol). The sheets were then washed three times with the same solution without calmodulin and after air-drying autoradiography was carried out using X-ray Kodak films. Overlay with [125I]actin was carried out as described above, but no CaC12 was added to the solution. For the visualization of the antibody reactions, the nitrocellulose sheets were blocked as described above and subsequently incubated 1 h with 2% milk p o w d e r / P B S containing the anti-spectrin antibodies in a 1 : 200 dilution. After washing with same solution without the antibodies, the sheets were incubated with a peroxidase-labelled second antibody (1 : 800) for 1 h. The reaction was then visualized with 4-chloro-l-naphtol. CNBr digestion of the a-subunit of lioer spectrin The t~-subunit of spectrin was fragmented with CNBr essentially as previously described [19]. The protein purified by calmodulin-affinity chromatography was precipitated with 8% TCA (in the presence of deoxycholate) at 4 ° C , washed once with acetone/HC1 and once with cyclohexane. The precipitate was solublized
by sonication in 70% formic acid, and digested with CNBr, under nitrogen, for 24 h at room temperature in the dark. The formic acid was evaporated at 4 5 ° C under nitrogen and the digest was washed twice with 300/~1 of water and finally suspended in electrophoresis sample buffer. The ~t-subunit obtained by electroelution was treated in the same way except that after removal of SDS the protein was directly solubilized in 70% formic acid.
Proteolysis in situ Proteolytic fragmentation of the a-subunit of spect i n has been carried out in situ using a procedure slightly modified from previously published methods [20]. a-Subunit-containing gel slices obtained by preparative gel electrophoresis were loaded on tricine buffer gels and overlayed with 70 /zl 'slice buffer' (50 mM Tris-HC1, 20% glycerol, 0.1% SDS (final p H 6.8)) containing different concentrations of S. aureus V-8 proteinase. After 15 min incubation, electrophoresis (at 70 V) was carried out until the sample reached the stacking gel/running gel interface and was then stopped for 15 min to allow protein digestion. Electrophoresis was then continued again overnight at 30 V. Production of anti-spectrin antibodies In this study the antigen was an a-spectrin which was freed from contaminants using preparative SDS-PAGE. The band was then cut out and the protein electroeluted. The purified protein (0.4 mg) was emulsified in 0.4 ml complete Freund's adjuvant, and intramuscularly injected into the back of several rabbits. A second injection was given 10 days later. Subcutaneous booster injections (0.4 mg a-spectrin in incomplete Freund's adjuvant) were given 6 weeks later. 10 days after the booster injections the rabbits were bled. More blood was then withdrawn at three day intervals. Immunocytochemical oisualization of a-spectrin Pieces of liver were rapidly frozen in liquid nitrogen and 4 /~m sections were cut using a cryotom. The sections were incubated for 30 min in PBS containing normal porcine serum (1:10), new born calf serum (1:10) and 0.01% sodium azide. After draining, the sections were incubated at room temperature for 1 h with the anti-a-spectrin antibodies diluted 1 : 40 in PBS containing new born calf serum (1:10) and 0.01% sodium. After washing with PBS (three times 10 min) the second antibody (porcine anti-rabbit diluted 1 : 80) was applied for 1 h. Finally, the sections were incubated for 1 h with rabbit peroxidase anti-peroxidase (PAP) diluted 1:80. To visualize the antibody reaction, the sections were incubated with diaminobenzidine (25 m g / m l in PBS (pH 7.6) containing 0.03% hydrogen peroxide).
76
Other methods Protein determination was carried out using a modified Lowry procedure [21]. Results
Localization of spectrin in the different functional domains of liver plasma membranes Spectrin present in rat liver subplasmamembrane fractions enriched in the different functional domains has been identified either by looking at the presence of the high molecular mass doublet after separation of the proteins by SDS-polyacrylamide gel electrophoresis or by [t25I]calmodulin overlay of the blotted proteins to visualize the a-subunit (Fig. 1, A and B). The results show that spectrin is mainly associated with the basolateral and the heavy basolateral fractions, and very little is present in the canalicular membrane fraction. A possible explanation for the lack of spectrin in the canalicular membrane is that this protein dissociates during the preparation of the membrane fractions. Indeed, we have observed that the membrane skeleton can easily dissociate from the plasma membrane depending on the medium used during the preparation. The spectrin which dissociates from the plasma membrane during homogenization can be recovered in the soluble fraction obtained after sedimenting the microsomes. For instance, the hypotonic bicarbonate-medium which is commonly used for the isolation of liver plasma membranes gives membranes which are almost devoid of spectrin (data not shown). On the other hand, the magnesium-containing isotonic medium that we have used in these experiments seems to be particularly suita-
ble in preventing dissociation of the membrane cytoskeleton. To further investigate the distribution of spectrin in the plasma membrane we have prepared antibodies against the a-subunit of liver spectrin and carried out an immunohistochemistry study. Immunoblotting experiments using either the whole plasma membrane or the isolated a-subunit clearly show that the antibodies reacted specifically with the a-subunit (Fig. 2). The immunohistochemical results shown in Fig. 3A seem to indicate that the antibodies react with the whole plasma membrane, including the canalicular region. No staining was observed in the control incubated with the preimmune serum from the same rabbit (Fig. 3B). Additional immunohistochemical studies carried out with affinity purified antibodies confirmed these results. The anti-aspectrin antibodies seem to react also with the nuclear envelope confirming a previously published observation which showed the presence of spectrin-like proteins in the nucleus [22].
Isolation of rat liver spectrin and separation of the two subunits We have shown that liver plasma membranes prepared in isotonic medium still retain most of their cytoskeleton. Five major components of this membrane associated cytoskeleton have been previously identified [23]. The first two are two polypeptides (52 and 56 kDa) which compose the intermediate filaments associated with desmosomes, the third is actin (43 kDa) and the last two are the two high molecular mass polypeptides which constitute spectrin (240 and 235 kDa). These proteins can also be recognized in the gel electrophoresis protein pattern of our liver plasma membrane (Fig.
A
B
kDa 200 116 97 66
45
0
1
2
3
4
1
2
3
4
Fig. 1. SDS/(4-12q$)-PAGE and [1251]calmodulin overlay of subplasmamembrane fractions. (A) SDS-PAGE pattern of membrane proteins (70 #g). Lane 1, whole plasma membrane; lane 2, bile canalicular membranes; lane 3, basolateral membranes; lane 4, heavy basolateral membranes; and lane 0, molecular mass standards from Bio-Rad. (B) Autoradiogram of the gel in A after [125I]calmodulin overlay.
77
kDa 240
were separated by gel permeation chromatography at high ionic strength and the fractions containing the partially purified spectrin were collected (Fig. 4, lane 6). The contaminants still associated with spectrin could be finally removed by centrifugation following dialysis against low ionic strength buffer. The two subunits of spectrin were dissociated by partial denaturation at a high concentration of sodium iodine, and the c~-subunit separated by means of calmodulin-affinity chromatography (Fig. 4, lanes 8 and 9). As shown in Fig. 5, the isolated a-subunit still maintains its ability to bind calmodulin and actin. The binding of [12SI]calmodulin is calcium-dependent and was not prevented by preincubating with a large amount of cold actin. Similarly, [125I]actin binding was not prevented by preincubating with cold calmodulin (data not shown) indicating that the two proteins do not bind to the same site.
~--
1
2
Fig. 2. Immunoblotting using anti-a-spectrin antibodies. Lane 1, whole plasma membranes; and lane 2, purified a-subunit of spectrin. The experimentalconditionsare describedin 'Materials and Methods'.
4, lane 1). In order to purify liver spectrin the basolateral membranes were first solubilized with Triton X-100 to remove most of the integral proteins. Actin and about 50% of spectrin could then be solubilized from Triton X-100 insoluble pellet by extraction with a high ionic strength alkaline medium (Fig. 4, lane 5). Most of the intermediate filaments remained in the pellet. Actin and spectrin present in the alkaline extract
Chemical and proteolytical fragmentation of the a-subunit of liver spectrin The location of the calmodulin- and actin-binding domains in the a-subunit of spectrin has been investigated by specific cleavage of the molecule either by chemical methods or by the use of proteinases. Cleavage at methionine residues with CNBr, at low concentration, gives a major calmodulin-binding fragment of 150 kDa (data not shown). At higher CNBr concentration a large number of fragments with a molecular mass below 66 kDa are generated (Fig. 6A). Several of these fragments still bind calmodulin, however, particularly a strong reaction was observed with fragments of 34, 32
~iii ~i
~i!i~i~i~iii!iiiii~i !iii ~i!iiiiiii;¸~i;i
Fig. 3. Immunohistochemistryof rat liver sections. The sections were prepared as described in 'Materials and Methods' and stained with either anti-a-spectrin antibodies (A) or preimmune serum (B). The scale bar corresponds to 10/xm.
78
iiiiiiiiiiiiii!iiiiiiiil;~iil
mm
0
1
2
34
5
6
7
89
Fig. 4. SDS/(4-I2~)-PAGE of the different fractions (70/~g) obtained during the purification of liver spectrin and the isolation of the a-subunit. Lane 1, whole plasmamembrane; lane 2, Triton X-100 supernatant; lane 3, Triton X-100 pellet; lane 4, alkaline pellet; lane 5, alkaline extract; lane 6, spectrin after the two gel filtration steps; lane 7, purified spectrin; lane 8, spectrin a-subunit; lane 9, spectrin fl-subunit; and lane 0, molecular mass standards.
and 23 kDa (Fig. 6B). At lower amounts of CNBr the 34 and 32 kDa fragments are better resolved (Fig. 7A), but no 23 kDa fragment is generated. Fig. 7 seems to indicate that the fragments which bind calmodulin can also bind actin. However, the presence of different fragments migrating in the same position can not be excluded. Calmodulin- and actin-binding fragments
A
B
having different molecular masses can be obtained by proteolysis of the a-spectrin with S. aureus V-8 proteinase. As shown in Fig. 8A, lane 1, if the digestion is carried out with a low amount of proteinase several calmodulin-binding fragments are generated, the smallest having a molecular mass of about 60 kDa. A 10-fold increase in the amount of proteinase causes the disappearance of many of the high molecular mass calmodulin-binding fragments, and the formation of a new calmodulin-binding fragment of 27 kDa (Fig. 8A, lane 2) and of an actin-binding fragment of 31 kDa (Fig. 8B, lane 2, indicated by the arrow). Discussion
Fig. 5. Autoradiograms of [12SI]calmodulin (A) and [125I]actin (B) overlays of the a-subunit of liver spectrin isolated by calmodulin-affinity chromatography.
Liver plasma membrane is a highly specialized membrane which contains three different functional domains. The membrane cytoskeleton which has been found associated with these domains has different functional and structural properties [24]. The immunocytochemical studies reported here show that spectrin is a common component of the membrane skeleton since it is uniformly distributed along the whole plasma membrane of hepatocytes. However, the way spectrin interacts with the membrane skeleton in the functional domains might be different. In fact, we have observed that sp.ectrin associated with the canalicular domain is easily released during the isolation of the subplasmamembrane fractions, whereas most of the basolateral
79
A
B
A
B
9~
61 4,' 3; .
1 Fig. 6. SDS-PAGE and calmodulin overlay of CNBr fragments of the a-subunit. (A) Right lane, Coomassie blue staining of the fragments obtained by CNBr digestion of the a-subunit using 3 #g CNBr per #g protein. The fragments were separated on 16.5% Tricine gels. Left lane, molecular mass standards. (B) Autoradiogram of the [12sI]calmodulin overlay carried out with the same CNBr fragments used in A. The left lane shows 14C-labelled low molecular mass standards (92, 55, 46, 30 and 14 kDa).
A
B
k Da 92-. 46-30-.
14,,-
¸I¸
i ¸¸
Fig. 7. Calmodulin and actin overlay of CNBr fragments obtained from the a-subunit of spectrin. Digestion was carried out at 1.6 pg CNBr per #g protein. (A) [12Sl]calmodulin overlay. (B) [l:Sl]actin overlay.
2
1
2
Fig. 8. 'In situ' proteolysis of the a-subunit of spectrin with S. a u r e u s V-8 proteinase. Gel slices containing about 40 pg of a-subunit were loaded on the gel and digested with either 70 ng V-8 proteinase (lane 1) or 700 ng V-8 proteinase (lane 2). (A) [125I]calmodulinoverlay. (B) [125I]actin overlay.
spectrin remains tightly b o u n d to the membrane. The basolateral spectrin can be extracted f r o m the membrane by alkaline treatment at high ionic strength. Using this procedure only half of the b o u n d spectrin is released, indicating that also in this m e m b r a n e different types of spectrin association might occur. A n interesting possibility which we are currently investigating is the existence of different isoforms of spectrin associated with the plasma m e m b r a n e of hepatocytes. Spectrin isoforms have already been f o u n d in other epithelial tissues and in brain [25,26]. Liver spectrin has been purified from the alkaline extract of basolateral m e m branes. The major c o n t a m i n a n t s f o u n d associated with spectrin are actin and a 200 k D a protein. The 200 k D a protein is tightly associated with spectrin and can only be removed in the last purification step since it gives insoluble aggregates when dialyzed against a low ionic strength buffer. Most likely this protein is analogous to red blood cell ankyrin, a protein involved in the binding of spectrin to the membrane. The a-subunit of liver spectrin still retains its ability to bind b o t h actin and calmodulin and can therefore be easily purified by calmodulin-affinity c h r o m a t o g r a p h y after partial denaturation of spectrin to allow dissociation of the two subunits. The a-subunit plays an i m p o r t a n t role in the regulation of spectrin function since it contains the binding site for calmodulin and actin. The localization of the calmodulin-binding site in the a-subunit is still a matter of debate, since different calmodulin-binding regions have been p r o p o s e d [6-10]. These different find-
80 ings could be explained by the presence of more than one binding site for calmodulin as it has been shown with caldesmon, another cytoskeletal calmodulin and actin-binding protein which binds two molecules of calmodulin [27]. However, the available studies indicate that the ct-subunit of brain can only bind one molecule [28]. Our studies on the identification of the calmodulin- and actin-binding site of the a-subunit were mainly directed towards the isolation of small calmodulin- and actin-binding fragments which could then be sequenced. The digestion of the a-subunit with high concentrations of S. aureus V-8 proteinase separates the two binding domains generating a 31 kDa fragfiaent which has lost the ability to bind calmodulin anda 27 kDa fragment which binds exclusively calmodulin. On the other hand, the digestion of the a-subunit with CNBr gives several fragments with a molecular mass of around 30 kDa which bind calmodulin a n d / o r actin. Whether the calmodulin and actin domains are localized on the same CNBr fragments can not be decided on the basis of our experiments. To get a conclusive answer a more detailed investigation using two-dimensional gel electrophoresis is needed.
Acknowledgements We wish to thank Prof. E. Carafoli for encouragement and helpful discussions. This work was supported by the Swiss National fond (Grant No. 3.531.-0.86).
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