Cellular localization of cytochrome css3 in the Nz-fixing cyanobacterium Anabaena variabilis. Aurelio Serrano*, Patricia Gim~nez, Siegfried Scherer, and Peter ...
Arch Microbiol(1990) 154:614- 618
Archwes of
Hicrobiology
9 Sprmger-Verlag 1990
Cellular localization of cytochrome css3 in the Nz-fixing cyanobacterium Anabaena variabilis Aurelio Serrano*, Patricia Gim~nez, Siegfried Scherer, and Peter Biiger Lehrstuhl fiir Physiologieund Biochemieder Pflanzen,UniversitfitKonstanz,Postfach 5560, W-7750 Konstanz, Federal Republic of Germany Received January 16, 1990/AcceptedJuly 24, 1990
Abstract. The "in situ" location of the electron carrier protein cytochrome c553 (cyt cs53) has been investigated in both vegetative cells and heterocysts of the cyanobacterium Anabaena variabilis ATCC 29413 using the antibody-gold technique, carried out as a postembedding immunoelectron microscopy procedure. When using a rabbit polyclonal anti-cyt c553 specific antiserum an intense labelling, associated mainly with the cell periphery (cytoplasmic membrane and periplasmic area), was seen in both heterocysts and vegetative cells. The selective release of most of the cellular cyt c553 during a Tris-EDTA treatment confirms a periplasmic localization of this protein in A. variabiIis. The results indicate that most of cyt c553 is located in the periplasmic space. The roles ascribed to this protein in both respiration and photosynthesis in cyanobacteria are discussed.
Key words: Anabaena -
Electron transport chain Cytochrome cs53- Photosynthesis - Respiration Protein localization - Immunogold labelling - Bluegreen algae
In cyanobacteria, respiratory electron transport has been claimed to share some components with photosynthetic electron transport (Peschek 1987; Scherer et al. 1988a). Among others, cytochrome c553 (cyt cs53) (Stfirzl et al. 1982; Alpes et al. 1984) has been suggested to be a component of both photosynthetic and respiratory electron transport chains in cyanobacteria. In membrane * Recipient of a Research Fellowship of the Alexander von Humboldt Foundation (Bonn, FRG) for a leave to the University of Konstanz. Permanent address: Instituto de Bioquimica Vegetal y Fotoslntesis, CSIC y Universidad de Sevilla, Apdo. 1113, 41080Sevilla, SPAIN. Offprint requests to: any author Abbreviations: Cyt c553, cytochrome c553; PBS, phosphate buffered
saline (20 mM sodium phosphate, 0.9% NaC1, pH 7.4); PMSF, phenylmethylsulfonylfluoride
preparations of Anabaena variabilis both cyt c5s3 (from the same species) and horse heart cyt c effectively donate electrons to P7oo (in the light) and cytochrome oxidase (Lockau 1981). Using membrane preparations of Anabaena sp. 7119, Stfirzl et al. (1982) restored electron flow from water to NADP + in the light, and from NAD(P)H to Oz in the dark, by adding purified Anabaena sp. 7119 cyt c553, which accepted electrons from the cytochrome b6/fcomplex. Since an antibody against this cyt c553 inhibited not only photosynthetic but also respiratory electron transport in these membrane preparations (Alpes et al. 1984), this protein may represent the native donor for cytochrome oxidase. In spite of the information available on the redox role of cyt c5s3 in cyanobacteria, direct experimental evidence on its actual cellular localization is still scarce. While it is clear that common links exist between photosynthesis and respiration and photosynthetic electron transport is located exclusively in the thylakoids, there is still some controversy concerning the site of respiratory electron transport in cyanobacteria. Cyanobacterial thylakoids exhibit repiratory activity (Peschek 1987; Scherer et al. 1988a) and, in fact, a cytochrome-c oxidase has been found in thylakoid membranes in both vegetative cells (Wastyn et al. 1988) and heterocysts (Hfifele et al. 1988; Wastyn et al. 1988) of A. variabilis. Nevertheless, recent reports on the presence of an aa3type cytochrome-c oxidase in the cytoplasmic membrane of both unicellular (Anacystis nidulans) (Molitor et al. 1987) and filamentous (A. variabilis) (Wastyn et al. 1988) cyanobacteria indicate that at least part of the respiratory chain is located also in this membrane. The in situ cellular location as well as selective solubilization by a Tris-EDTA treatment reported in this paper provide direct evidence for the localization of cyt c553.
Materials and methods Organism and growth Anabaena variabilisATCC29413 was grownunderdinitrogen-fixmg conditmns at 30~ in continuous light as described (Sandmann
615 1986) and harvested 2 days after inoculation (late logarithmic phase). Chlorophyll a was determined in methanol at 665 nm (Scherer et al. 1988 b). Protein content was measured using the BioRad (Munich, FRG) protein assay.
Antibody preparation A polyclonal antibody was raised in rabbits (Alpes et al. 1984) against cyt c553 purified to homogeneity from Anabaena sp. strain 71 t9. The antiserum was used either directly or after ammonium sulfate fractionation (Lwingston 1974) with similar results. Rabbit preimmune serum was used as a control for immunocytochemical experiments.
(Siegelman and Kycia 1978). For cyt c5s3 determination the supernatans were treated with ammonium sulfate at 70 % saturation (15 min, 4 ~C), taking advantage of the fact that the cyanobacterial soluble cytochromes of c-type remain in solution after this treatment (Matsubara and Wada 1988). Cyt c553 was contained in its reduced form in the supernatant fraction after centrifugation (10000g, 10min) and remained reduced when kept on ice until its spectrophotometric determination using the AA553 580 (Morton 1975; Stiirzl et al. 1982). Untreated A. variabihscells were disrupted by three freeze-thawing cycles followed by ultrasonic treatment at 4~ and eventually used to estimate the total amount of cellular soluble protein, C-phycocyanin and cyt cs53. All absorbance measurements were performed at room temperature m a Shimadzu UV 369 recording spectrophotometer.
Western blotting
Chemicals
Cyt cs53 from A. variabilis was detected immunologically, either in purified preparations or in cell-free extracts, after SDS-PAGE (15 % acrylamide) and transfer of the proteins to nitrocellulose. The antigen was detected by using the antibody in a 1 : 1000 dilution with a peroxidase-conjugated goat anti-rabbit IgG (Sigma, Munich, FRG) as the secondary antibody (Towbin et al. 1979). Cross-reactivity of the antibody against Anabaena ap. 7119 cyt c553 with the same protein from A. variabilis and specificity of the antiserum were demonstrated in both crude extracts and purified cyt c553 preparations.
All chemicals used were of analytical grade and have been purchased from Sigma (Munich, FRG), Merck (Darmstadt, FRG) or Roth (Stuttgart, FRG).
Fixation, embedding and immunocytochemical labelling A. vanabilis filaments were fixed with 2.5 % (w/v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 2 h at 4~ dehydrated in ethanol (30 - 100 %) and twice in propylene oxide, and embedded in resin Epon 812 (Polysciences, USA). Ultrathin sections, obtained on a Reichert ultramicrotome using glass knives, were mounted on formvar-coated gold or nickel grids (200 mesh). The sections were etched in a 10% (v/v) aqueous solution of H2Oz for I0 min; incubated for 10 rain with NaBH4 (3 mg/ml) m distilled water; washed in PBS; incubated for 1 h in PBS supplemented with 5% newborn calf serum, 0.2% Tween 20 and 0.5 M NaC1 (PBS/ST): incubated for 2 h in the antiserum diluted 1 : 10 or 1 : 20 in PBS/ST: washed in PBS/ST: incubated for 2 h in goat anti-rabbit IgG conjugated with 10 nm gold particles (Janssen Pharmaceutica, Belgium) diluted 1:20 (v/v) in PBS/ST; washed in PBS/ST and m distilled water, and finally air-dried. Sections were stained with 4% aqueous uranyl acetate for 2 h and alcaline lead citrate (Reynolds 1963) for 5 min. The sections were studied with a Zeiss 9S TEM operating at an acceleration voltage of 60 kV. The distance from the antigen to the gold marker - estimated from the size of the antigen-antibodies complex (Rohde et al. 1988) - was considered to be 40 nm, being used as the maximal tolerable distance of a gold particle to be counted as a label for a given cell structure.
Tris-EDTA treatment The cells were harvested, washed twice m 50 mM Tris-HC1, pH 8, 30 mM NaC1 by centrifugation (10000 g, 5 rain) at room temperature, and the resulting pellet was resuspended, at a chlorophyll concentration of 0 . 8 - 1 ~tmol/ml, in the same buffer supplemented with 40 mM EDTA - Na2, 0.5 M (or 0.75 M) sucrose, 2 mM PMFS and 5 mM e-aminocaproic acid. During incubation with gentle agitation at 30~ aliquots were withdrawn, the cells were removed by rapid centrifugation (10000g, 1 rain), and the supernatans were used for both protein measurements and spectrophotometric estimation of C-phycocyanin. The AA62o_73o was used to estimate the relative amounts of this chromoprotein in the supernatants
Results and Discussion T h e specific a n d a c c u r a t e l o c a l i z a t i o n o f e n z y m e s is o f special i m p o r t a n c e in p r o k a r y o t e s b e c a u s e a full s p e c t r u m o f m e t a b o l i c activities is c a r r i e d o u t in a n u n c o m p a r t m e n t a l i z e d cell. T h e cellular l o c a l i z a t i o n in c y a n o b a c t e r i a l t h y l a k o i d m e m b r a n e s o f extrinsic p r o t e i n s t h a t p a r t i c i p a t e in b o t h p h o t o s y n t h e t i c a n d r e s p i r a t o r y e l e c t r o n t r a n s p o r t chains, i.e. f e r r e d o x i n - N A D P + o x i d o r e d u c t a s e ( S e r r a n o et al. 1986; Scherer et al. 1988 b), has been a c h i e v e d b y using gentle cell f r a c t i o n a t i o n m e t h o d s . This is a difficult t a s k with p r o t e i n s w h i c h interact o n l y very w e a k l y with m e m b r a n e s , as it is the case o f c y t c553. T h e r e f o r e , we h a v e i n v e s t i g a t e d t h e "in situ" cellular l o c a t i o n o f the e l e c t r o n c a r r i e r p r o t e i n cyt c553 in cells o f the c y a n o b a c t e r i u m A. variabilis using the antib o d y g o l d t e c h n i q u e c a r r i e d o u t as a p o s t - e m b e d d i n g immunoelectron microscopy procedure. Colloidal gold particles have been used successfully for the i m m u n o c y t o c h e m i c a l l o c a l i z a t i o n o f a n u m b e r o f p r o t e i n s in c y a n o b a c t e r i a l cells w h e n u s i n g c o n v e n t i o n a l E p o n e m b e d d i n g ( B e r g m a n et al. 1985; B r a u n - H o w l a n d et al. 1988; L i n d b l a d 1989; L i n d b l a d a n d B e r g m a n 1989). A s was e x p e c t e d f r o m their very similar p h y s i c o c h e m i c a l p r o p e r t i e s ( B 6 h m e a n d Pelzer 1982), W e s t e r n b l o t t i n g a n a l y s i s i n d i c a t e d t h a t t h e a n t i b o d y raised a g a i n s t the cyt c553 o f Anabaena sp. strain 7119 s t r o n g l y c r o s s - r e a c t e d w i t h the s a m e h e m o p r o t e i n f r o m A. variabilis. T h e results o f the i m m u n o g o l d labelling experiments in the two cell types, n a m e l y v e g e t a t i v e cells a n d heterocysts, p r e s e n t in N z - f i x i n g A. variabilis f i l a m e n t s are s h o w n in Fig. 1. G o l d - l a b e l l i n g o f the c y t css3, o b t a i n e d b y t r e a t i n g thin sections w i t h the r a b b i t antis e r u m specific for this p r o t e i n , resulted in a p r e f e r e n t i a l scattering o f g o l d particles in the p e r i p h e r i c regions (cytop l a s m i c m e m b r a n e a n d p e r i p l a s m i c area) o f b o t h v e g e t a t ive cells (Fig. I A ) a n d h e t e r o c y s t s (Fig. 1B), the p e r i p h e r i c label d i s t r i b u t i o n b e i n g p a r t i c u l a r l y clear in these N2-fixing cells. T h e e x t e r n a l c y t o p l a s m i c region, in w h i c h m o s t o f c o n v o l u t e d t h y l a k o i d s a r e l o c a t e d in
616
Fig. 1 A-C. Immunocytochemical localization of cyt c553in vegetative cells (A) and heterocysts (B) of A. variabilis. CE, cell envelope layers (cell wall and cytoplasmic membrane); T, thylakoid membranes; IB, inclusion body; P, heterocystous plug. Note that labelling is preferentially located at the cell periphery, near the cytoplasmic membrane and the periplasmic area (arrows). No labelling is observed when rabbit preimmune serum was used (C). The calibration bar represents 0.5 ~tm
~- lOO] ._o 75
#
25
I [-41 0
,
C
THY CY
,z
IB
Fig. 2. Distribution of coloidal gold particles in the localization of cyt c553 in A. variabilis vegetative cells by immunoelectromicroscopy. 21 individual cells were examined. In control experiments; the number of gold particles on cell sections varied between zero and 0.6% of the numbers of particles associated with antiserum treated sections. C, cytoplasmic membrane and periplasmic regions; THY, intracellular thylakoid membranes; CK cytoplasm; IB, inclusion bodies (i. e., carboxysomes). The maximal tolerable distance of a gold particle from the analyzed cell component to be counted as a label for the respecting cell structure was considered to be 40 nm. Marker bars represent SD
Anabaena species (Startler 1988), appears also labelled in vegetative cells. In contrast, only a few gold particles are observed in the central region, the "centroplasm", which contains the genetic material. Non-specific background labelling on the formvar film was very low and, in accordance with this fact, labelling is absent in control experiments when rabbit preimmune serum was used (Figs. 1 C). The percentage distribution of gold particles obtained in thin sections of A. variabilis vegetative cells is shown in Fig. 2. Statistical data clearly indicate that, in fact, label located at or near the cytoplasmic membrane
encounted for 7 0 - 7 5 % of the total number of gold particles per cell. It is well established that all the low-molecular mass bacterial cytochromes of c-type so far studied are located in the periplasmic region, either in the periplasmic space or on the periplasmic side of the membrane (Ferguson 1988; Pettigrew and Moore 1987). We treated A. variabilis cell suspensions with Tris plus EDTA, checking for a selective release of a periplasmic cyanobacterial cyt c553This release during such a treatment is a well-established criterion to demonstrate a periplasmic location of proteins in gram-negative bacteria (Garrad 1971; Ferguson 1988). Figure 3 shows the release of C-phycocyanin, which can be considered as a marker for cytoplasmic proteins in cyanobacteria, soluble protein and cyt c553 by A. variabilis cell suspensions in the presence of Tris, EDTA and two different concentrations of the osmotic stabilizer sucrose. The selective release of most of the cytochrome c without a massive liberation of cytoplasmic contents is demonstrated, especially when the higher concentration of sucrose is used (see Fig. 3 B). The presence of cyt c553 in the protein fractions released by Tris-EDTA treatment was confirmed by Western blotting analysis: a single polypeptide, exhibiting the same molecular mass (about 11000) as the purified A. variabilis hemoprotein, was recognized by the rabbit anti-cyt cs 53 antiserum (data not shown). These results indicate that most of cyt c553 has been effectively released before the cell integrity was severely damaged. In fact, large amounts of soluble cytochrome ( 6 0 - 70% of total cellular content) are liberated after 30 min of Tris-EDTA treatment, when only 1 - - 2 % of total soluble cellular protein and less than 1% of C-phycocyanin were released (see Fig. 3). These data point to a periplasmic location of the main part of the cellular content of this electron carrier protein in the cyanobacterium A. variabilis and are, therefore, in agree-
617
0o4t8]o
ill/ f~~.~.~B
0.03 J 6
1
0.02
0
30
60
TIME(rain)
J4
90
Fig. 3A, B. Rate of release of soluble cyt c5s3, esmnated by AAssz- 580, c-phycocyanin, estimated by AA6zo 73o, and protein during Tris-EDTA treatment of A. variabilis cell suspensions in the presence of 0.5 M (A) or 0.75 M (B) of the osmotic stabilizer sucrose. Details are in the text. In both cases, final release of cyt c553 represents 70-80% of the amount exhibited by a sonic extract of untreated cells
ment with the immunocytochemical results presented above. The periplasmic localization o f c y t c5 s 3 in A. variabilis cells was unexpected. The general view of this hemoprotein is that of an electron carrier o f the photosynthetic electron chain which is located exclusively in the intracellular thylakoid membranes. Nevertheless, it should be kept in mind that c-type cytochromes are found in the periplasm of all gram-negative bacteria so far studied (Ferguson 1988). Indeed, the cytochrome c2 of purple photosynthetic bacteria, which re-reduces the reaction center in the photosynthetic electron transport chain, is trapped in the periplasmic space in vivo (Prince et al. 1975). Our results suggest that a similar situation, but involving cyt C5sa and the respiratory rather than the photosynthetic electron chain, might be found in cyanobacteria. It should be noted that a variety of exogenous c-type cytochromes are oxidized effectively by intact spheroplasts of the unicellular cyanobacterium Anacystis nidulans (Peschek et al. 1982), and an aa3-type cytochrome oxidase has been found in the cytoplasmic m e m b r a n e of several cyanobacteria (Molitor et al. 1987; Wastyn et al. 1988). The periplasmic cyt c55a found in A. variabilis cells p r o b a b l y is the physiological electron donor for the cytochrome-c oxidase present in the cytoplasmic m e m b r a n e of this cyanobacterium. The reducing agent for periplasmic cyanobacterial cyt Cs53 remains to be identified but it is interesting to note that low-potential m e m b r a n e - b o u n d cytochromes, different from thylakoidal cytochromes, and N A D ( P ) H - c y t c reductase activities have been found in the cytoplasmic m e m b r a n e of Anabaena and Anacystis ( O m a t a and M u r a t a 1984, 1985).
Moreover, a fructose-dependent ferricyanide-reducing redox system that is also located in the cytoplasmic membrane has been reported in whole cells of several cyanobacteria (Scherer and B6ger 1985). On the other hand, the presence of periplasmic cyt Cs53 in heterocysts should stimulate further research on a possible role of this electron carrier protein in the so-called respiratory protection of the Oz-labile nitrogenase (e.g. Gallon 1981). This protection should be very efficient if the proteins involved are located at the cell periphery, i.e. in the periplasmic area. Acknowledgements. A.S. thanks the Alexander von Humboldt Foundation for support. The authors thank Prof. W. Rathmayer (University of Konstanz) for providing electron microscopy facilities. This work was supported by grants of the Deutsche Forschungsgemeinschaft.
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