Vol. 138, No. 1

JOURNAL OF BACTERIOLOGY, Apr. 1979, p. 207-217 0021-9193/79/04-0207/11$02.00/0

Modifiable Chromatophore Proteins in Photosynthetic Bacteria KAM M. HUIt AND RONALD E. HURLBERT* Department of Bacteriology and Public Health, Washington State University, Pullman, Washington 99164 Received for publication 10 February 1979

The chromatophores of Chromatium vinosum, as well as six other photosynthetic bacteria, contained two or more proteins which were insoluble when heated in the presence of sodium dodecyl sulfate (SDS) and 2-mercaptoethanol (,8-ME). When the chromatophores were dissolved at room temperature in SDS-,B-ME, these proteins were present in the SDS-polyacrylamide gel electrophoresis profiles, but when the samples were dissolved at 1000C, they were absent or considerably diminished. When one-dimensional gels of chromatophores solubilized at room temperature were soaked in the SDS-,B-ME solution and heated to 1000C and the gels were run in a second dimension, the proteins became immobilized in the original first-dimension gel, where they could be detected by staining. The two major proteins so affected in C. vinosum had apparent molecular weights of 28,000 and 21,000. The chromatophores of several other photosynthetic bacteria also contained predominant proteins between 30,000 and 19,000 molecular weight, which became insoluble when heated in the presence of SDS and ,8ME. In at least two of the species examined, these appeared to be reaction center proteins. The conditions causing the proteins to become insoluble were complex and involved temperature, SDS concentration, and the presence of sulfhydryl reagents. The chromatophores of four of the Chromatiaceae species and two strains of one of the Rhodospirillaceae species examined had a protein-pigment complex that was visible in SDS-polyacrylamide gel profiles of samples dissolved at room temperature but was absent in samples dissolved at 1000C.

amide gel electrophoresis (SDS-PAGE). Chromatophores from Rhodopseudomonas sphaeroides (3, 16, 24, 37, 39, 40), Rhodospirillum rubrum (7, 41), Rhodopseudomonas capsulata (9, 25), and Chromatium vinosum (14, 17, 23) have all been shown to contain dominant proteins in the 10,000- to 40,000-molecular-weight range. The isolated reaction centers of Rp. capsulata (25, 26), Rp. sphaeroides (2, 3, 31, 39, 40), Rs. rubrum (42), and C. vinosum (14,23) contain three predominant proteins with molecular weights of approximately 28,000, 24,000, and 21,000. The light-harvesting bacteriochlorophyll of these species is associated with proteins of about 10 kdaltons (2, 12, 27, 32). Some of the dominant chromatophore proteins are not seen in SDS-PAGE gels under certain conditions of solubilization. Clayton and Haselkorn (3) and Takemoto and Lascelles (40) reported that the 19- and 22-kdalton proteins of Rp. sphaeroides reaction centers are lost when the samples are boiled longer than 1 min in the solution used to dissolve the membranes before SDS-PAGE t Present address: Department of Microbiology and Immunology, Northwestem University Medical School, Chicago, analysis. Concomitant with this loss is an increase in accumulation of nonmigrating material IL 60611.

The photochemical activities of photosynthetic bacteria are localized in intracytoplasmic membranes (6, 17, 29, 38). When photosynthetic bacteria are disrupted, these membranes are converted into vesicles, terned chromatophores, which are usually isolated by a combination of differential and gradient centrifugations (5, 7, 11, 15, 19, 20). Chromatophores exhibit most of the light reactions associated with photosynthesis and have been shown to contain two pigment complexes: the light-harvesting, or bulk, bacteriochlorophyll; and the reaction center or phototrap pigments (22, 40). These complexes consist of bacteriochlorophyll, carotenoids, and accessory components associated with different proteins in special physical environments in the membrane (1, 32, 40). Recently, attention has turned to the protein components of the chromatophores. Isolated chromatophores and pigment complexes from a number of photosynthetic bacteria have been analyzed by sodium dodecyl sulfate-polyacryl-

207

208

HUI AND HURLBERT

J. BACTERIOL.

phosphate buffer (pH 7.5)-10 mM MgCl2 was employed. The results were similar with both buffers. The T. roseopersicina and A. roseus cells suspended in their respective growth media were thawed and disrupted as described above. In all cases, small quantities of RNase and DNase were added to the suspension before breakage. All extracts were centrifuged at 17,300 x g for 20 min at 40C to sediment whole cells and debris. Chromatophores of C. vinosum were isolated from the supernatant by flotation (43), isopycnic gradient centrifugation (17), or differential centrifugation. In the third case, the chromatophores were sedimented at 140,000 x g for 1 h, and the upper deepred portion of the pellet (approximately 75% of the material) was carefully scraped off with a spatula and suspended in distilled water. This procedure was repeated two more times. The protein banding patterns of chromatophores isolated by all three procedures were indistinguishable; all of the preparations contained some cell wall material. Since the differential centrifugation procedure was simpler, all further chromatophores were isolated by this technique. Ribosomes were isolated as described by Mechler and Oelze (23). C. vinosum cell walls were isolated by nisms were used in this study: C. vinosum (formerly isopycnic gradient centrifugation (17) or by a modifistrain D), Rp. capsulata; Rp. palustris Kl and 42 (10); cation of the Triton X-100 method of Schnaitman (35; Rp. sphaeroides L; Thiocapsa roseopersicina; Thio- B. Lane and R. E. Hurlbert, unpublished data). Both cystis violacea 2311; and Amoebobacter roseus 6611. preparations were free from significant intracytoThe Rp. capsulata and Rp. palustris strains were gifts plasmic membrane as indicated by their low bacterof G. Drews, Biologisches Institut II, University of iochlorophyll content. Both preparations gave identiFreiburg, Freiburg, Germany. Rp. sphaeroides was cal SDS-PAGE patterns when stained for proteins or obtained from J. Lascelles, Department of Microbiol- lipopolysaccharide (17). When French press extracts ogy, University of California, Los Angeles. The cul- of T. roseopersicina, A. roseus, T. violacea, Rp. captures of T. violacea and A. roseus were provided by sulata, and Rp. sphaeroides were extracted with TriN. Pfenning, Institut fur Mikrobiologie, Gottingen, ton X-100 by the modified method of Schnaitman (35), all produced insoluble fractions that (i) were low in Germany. The four Chromatiaceae species were all cultivated bacteriochlorophyll; (ii) contained large vesicles when on the mineral salts medium previously described (18). examined under an electron microscope; and (iii) had C. vino8um and T. roseopersicina were grown on 0.02 SDS-PAGE patterns markedly different from that of M DL-malate as the sole carbon source. T. violacea intracytoplasmic membrane isolated from the same was cultivated lithotrophically on 0.3% Na2S2O3-0.5% species. SDS-PAGE. All of the reagents for preparing and NaHCO3, and A. roseus was grown on 0.02 M sodium acetate-0.5% NaHCO3. Rp. sphaeroides was culti- running the SDS-PAGE were obtained from Sigma vated photoanaerobically in a medium containing Chemical Co. and were prepared by the method of 0.05% KH2PO4, 0.04% MgSO4, 0.04% NaCl, 0.05% King and Laemmli (21). The slab gels (10% acrylamNH4Cl, 0.005% CaCl2 2H20, 0.1% disodium succinate, ide) were cast between glass plates (12 by 14 cm, Bio0.1% yeast extract, 0.0005% iron citrate, and 1 ml of Rad model 220) to a height of 9.5 cm, using spacers trace salts solution (18) per liter. The remaining Rho- 0.75 or 1.5 mm thick as required. The gel solution was dospirillaceae species were grown photoanaerobically overlaid with water until polymerization was comon RAH medium (8). All cultures were harvested pleted. The water overlay was replaced with a solution during the stationary phase, and the cell paste was containing buffer and SDS at the same concentration stored at -20°C until needed. Because their density as that in the gel, and the slab was stored overnight at was similar to that of the medium, T. roseopersicina 70C. A stacking gel (3% acrylamide) was prepared so and A. roseus cells did not sediment completely, even as to give a 1-cm distance from the well bottom to the upon prolonged (1 h) centrifugation at 40,000 x g. top of the analytical gel. Samples were prepared by Therefore, these cells, suspended in one-half to one- mixing an equal volume of sample buffer (0.125 M third of their original volume of growth medium, were Tris-hydrochloride [pH 6.9], 4% SDS, 10% ,B-mercapfrozen at -20°C. toethanol [,E-ME], 20% glycerol, 0.002% bromophenol Preparation of chromatophores, ribo8omes, blue) with a sample containing 10 to 100 ,ug of protein. and cell walls. Where possible, the bacteria were All samples were prepared in tubes (50 by 5 mm) suspended in buffer (1-g frozen pellet in 2 ml of buffer) closed with cork stoppers. Stock commercially preand were ruptured by two passages through a French pared ,B-ME was used for the majority of the experipressure cell (Aminco) at 15,000 to 20,000 lb/in2. In ments, since it was found that samples prepared with most cases, the buffer used was 10 mM Tris-hydro- freshly distilled ,B-ME produced the same results. The chloride (pH 8.0); in some cases, 50 mM potassium results were the same whether glycerol was present

at the top of the gel. Shepherd and Kaplan (37) reported similar findings with Rp. sphaeroides. Mechler and Oelze (23) observed that the C. vinosum reaction center contained only one predominant protein when the sample was treated with sample buffer at 700C before electrophoresis, but when it was treated at 30°C three proteins with apparent molecular weights of 30,000, 25,000, and 19,000 were detected. During an investigation of the protein composition of chromatophores of C. vinosum, we noted that several major proteins were lost when the membranes were heated in the membranedissolving solution before SDS-PAGE and that the intensity of the material staining at the top of the gels increased. The following is a report of a study of these modifiable proteins in C. vinosum and similarly modifiable proteins in several other photosynthetic bacteria. MATERIALS AND METHODS Organisms and cultivation. The following orga-

VOL. 138, 1979

MODIFIABLE CHROMATOPHORE PROTEIN

during the heating or whether it was added afterwards. Electrophoresis was carried out at 7.5 mA per gel (0.75 mm thick) until the sample had completely entered the stacking gel and then at 20 mA per gel until the ion front reached the end of the gel. Flowing tap water was used for cooling. The slabs were washed by gentle shaking in acetic acid-methanol-deionized water (1:5:5, vol/vol/vol) for 1 to 2 h. The gels were stained with gentle shaking for 2 h in 1.0% Coomassie brilliant blue R-methanolwater (1:1, vol/vol) to which 0.7 ml of glacial acetic acid was added for every 10 ml of dye solution immediately before use. Destaining was achieved either by shaking the gels for 30 min in acetic acid-methanolwater (1:5:5, vol/vol/vol) followed by 1 to 3 h in 7% (vol/vol) acetic acid or by shaking overnight in 7% acetic acid. In both cases, a small bag of activated charcoal granules was included in the tray to absorb the free dye. For two-dimensional-modification studies, a modification of the Russell (34) procedure was used. Samples were dissolved in sample buffer and run in 0.75mm-thick slabs for the first dimension. Immediately after the slabs were removed, strips containing the samples were cut out and placed in 50-ml screwcapped test tubes containing the soaking reagent. In every case, the soaking reagent contained 10% glycerol and 0.0625 M Tris buffer (pH 6.9); SDS and ,B-ME were added as indicated. After 15 min, strips which were to be heated were transferred to tubes containing the soaking reagent equilibrated to the desired temperature, where they were incubated for the indicated time. Immediately after a particular treatment, the gel strip was laid across the top of a 1.5-mm-thick slab (containing 1.5 cm of stacking gel), and a 1% agarose solution in sample buffer (cooled to 45°C) was poured around it. As soon as the agar had solidified (within 2 min), electrophoresis was begun. Bromophenol blue (0.06 ml of a 0.025% solution) was added to the top buffer, and the amperage was doubled. After electrophoresis, both the one-dimension original gel strip and the second-dimension gel were stained as previously described.

RESULTS SDS-PAGE of C. vinosum chromatophore proteins under various solubilization conditions. Both solubilization conditions and the age of the chromatophores affected the appearance of the samples in SDS-polyacrylamide gels before and after staining. Before staining, several pigmented areas were visible in the SDS-polyacrylamide gels. With fresh chromatophores (those less than 4 to 6 weeks old) solubilized at low temperatures, a series of high-molecularweight red bands was seen at the tops of the analytical gels. These bands were not present in samples that had been repeatedly frozen and thawed for several months (aged samples), nor were they present in fresh samples that had been heated at 500C for 30 min or at 1000C for 1 to 5 min. They were present, although in diminished

209

amounts, in samples heated at 500C for 10 min. In addition, all samples solubilized at room temperature, 37°C, or 50°C had a broad red band with an apparent molecular weight of 69,000 (hereafter referred to as the 69K colored complex). When samples were heated at 1000C, the 69K colored complex disappeared, and the intensity of the pigmented material at the tops of the analytical gels increased. Some pigmented material ran ahead of the dye front under all conditions, but the intensity of the pigment migrating in this position was increased in samples heated at 1000C. These differences were enhanced by staining (Fig. 1). In addition, other changes were seen with fresh chromatophores; three bands with estimated molecular weights of 32,000 (32K), 26,000 (26K), and 21,000 (21K) all increased in intensity as the time and/or the temperature of incubation increased (up to 500C for 10 min) (Fig. 1). Aged chromatophores solubilized at room temperature produced the same pattern as fresh chromatophores heated at 500C for 10 min (Fig. 1, lane 10). When samples were heated to 1000C, the 21K band disappeared and the 26K band decreased in intensity, whereas the 32K band remained unchanged. Furthermore, bands of intensely staining material were seen at the tops of gels of samples heated to 1000C. Only one major additional protein, with an apparent molecular weight of 42,000, was seen in the 100°C-heated samples (Fig. 1). This protein has been shown to be the major protein present in C. vinosum cell walls (17). At temperatures below 600C, the cell wall solubilizes in the sample buffer to form a series of high-molecular-weight complexes (>70,000) that enter only the first 2 cm of the analytical gel. At higher temperatures, the cell wall proteins are converted to their monomers; however, none of the cell wall proteins runs in the same position as the chromatophore proteins discussed here (17, 23). Since the chromatophores isolated by differential centrifugation probably contain ribosomes, pure ribosomes were isolated and analyzed by SDSPAGE. None of the ribosomal proteins ran in the same positions as the predominant proteins discussed here, nor were any of them modified by the conditions used here (unpublished data). To investigate the factors affecting the solubility of chromatophore polypeptides, one-dimensional gels of chromatophore preparations solubilized at room temperature or at 500C for 10 min were subjected to a variety of treatments and run in a second dimension, using the procedure of Russell (34). When the one-dimension gels of room-temperature-solubilized chromatophores were soaked in sample buffer for 15 min at room temperature, the majority of the pig-

210

J. BACTrERIOL.

HUI AND HURLBERT

Is-HMWCA

1

69k-. -

2

3

4

5

6

7

8

9

10

I

32k-. 26k21 k.

LC -| FIG. 1. Coomassie brilliant blue-stained SDS-PAGE gel of C. vinosum chromatophores solubilized under various conditions. Lanes I through 3 were solubilized at room temperature for 5, 30, and 60 min, respectively. Lanes 4 and 5 were solubilized at 37°C for 10 and 60 min, respectively. Lanes 6 and 7 were solubilized at 50°C for 10 and 30 min, respectively. Lanes 8 and 9 were solubilized at 100°C for 1 and 5 min, respectively. Lanes 10 and 11 were solubilized at room temperature and 100°C, respectively, for 5 min. Freshly prepared chromatophore samples (95 jg ofprotein per well) were used in wells 1 through 9. An aged chromatophore sampkle (100 pg of protein) was used in wells 10 and 11. OM, Major outer membrane contaminating protein; HMWC, high-molecular-weight pigmented complexes; IS, insoluble material; LC, low-molecular-weight polypeptides and colored material running ahead of dye front. Predominant proteins: 32K, 26K, 21K, and 69K colored complex.

ments and proteins migrated out of the first dimension into the second and lay on a 450 line (Fig. 2A). Some of the high-molecular-weight red band material remained in the first-dimension gels, and a trace of pigment migrated with the dye front (Fig. 2A). All of the 32K, 26K, and 21K proteins were on the 450 line (Fig. 2A). When one-dimension gels of room-temperaturesolubilized chromatophores were soaked and heated at 1000C for 5 to 10 min in sample buffer, four Coomassie brilliant blue-staining bands failed to migrate out of the first dimension. These immobilized components included material at the tops of the gels, part of the 69K colored complex, part of the 26K protein, and all of the 21K protein (Fig. 2B). It thus seems possible that the bulk of the material seen at the tops of one-dimension gels of samples heated to 1000C is composed of these components. In addition, a protein with an estimated molecular weight, in the unheated samples, of 16,000 shifted to a position corresponding to a molecular weight of 25,000 (25K), and part of the 69K colored complex was converted to low-molecular-weight material which ran ahead of the dye

front (Fig. 2B). Furthermore, some of the highmolecular-weight material at the tops of the onedimension gels was solubilized by this treatment. One of the proteins seen in this portion of the second-dimensional gel corresponded to the 32K protein (Fig. 2B). When first-dimensional gels were heated at 600C instead of 100°C, all of the proteins migrated out of the first dimension and into the second; however, a series of aggregates of the 21K protein was formed, and part of the 69K colored complex was converted to low-molecular-weight material (Fig. 20). The material at the tops of the one-dimension gels solubilized to produce a series of spots, three of which corresponded to the 32K, 26K, and 21K proteins (Fig. 20). In addition, the 25K protein again appeared. When the one-dimension gels were soaked and heated to 1000C in buffer alone, the picture was qualitatively similar to that seen with sample buffer, except that only a small portion of the 26K and 21K proteins remained behind in the first-dimension gels. When the first-dimension gels were soaked and heated in 5% ,f-ME (sample buffer without SDS), not only did the 69K colored complex and 26K and 21K

VOL. 138, 1979

MODIFIABLE CHROMATOPHORE PROTEIN I4WC

211

26K 21K

I-cc

I

I

261 21x

3?Kl6K?IK13K

C L

O1 _-

........

.

-

21.k-

FIG. 2. Two-dimensional SDS-PAGE gels of C. vinosum chromatophores treated in various ways. Individual gel strips ofchromatophore material dissolved in sample buffer at room temperature for 5 min and run in the first dimension in SDS-PAGE were cut out and treated as follows before being subjected to seconddimension SDS-PAGE: (A) soaked in sample buffer for 15 min at room temperature; (B) soaked in sample buffer for 15 min, followed by heating 100°C for 5 min; (C) soaked as in (A), heated at 60°C for 20 min; (D) soaked in sample buffer without SDS for 15 min, heated at 100°C for 5 min. ICC, Insoluble colored complex; all other abbreviations are as for Fig. 1. The strips at the top of the gels in (A), (B), and (D) are the original first-dimension strips stained after the second-dimension run. Since the first-dimension strip ofgel C contained no stainable material, it was not included. (A through D) Positions of the 32K, 26K, and 21K proteins on the diagonal are located by arrows. (C) Aggregates of the 21K protein are indicated by large arrows on the right, and the 32K, 26K, and 21K proteins that originate from the HMWC are indicated by small arrows on the left of the gel. The gels in (A), (B), and (C) are not aligned exactly because of the difference in drying of the 0. 75mm one-dimension gels and the 1.5-mm two-dimension gels.

proteins become immobilized in the first dimension, but most of the 32K protein, as well as a protein with an estimated molecular weight of 13,000, became immobilized (Fig. 2D). When one-dimension gels were heated, after soaking, at 100°C in 2% SDS alone, only a small portion of the 69K colored complex was immobilized, and all of the other proteins migrated into the second-dimension gels; however, the 21K protein formed an aggregate series, and the 25K protein

appeared under these conditions; i.e., the appearance of the second-dimension gels was similar to that shown in Fig. 2C, except that some of the 69K colored complex remained fixed in the first-dimension gels. When one-dimension gels were first soaked in 2% SDS for 15 min and then heated at 1000C for 5 min, followed by a further soaking for 15 min in 5% ,B-ME without heating, no proteins were immobilized, but the 21K protein did form an

212

HUI AND HURLBERT

aggregate series in the second-dimension gels. However, when a duplicate strip was heated at 1000C for 5 min after the f8-ME soak or when the strip was first soaked and heated in 5% ,8ME followed by soaking and heating in SDS, the 69K colored complex and the 26K and 21K proteins were rendered immobile and remained in the first-dimension gels, as in Fig. 2B. In all of these cases, the 25K protein was seen. To determine whether the phenomenon was restricted to fl-ME alone or whether other sulfhydryl reagents induced similar effects, ,BME was replaced with equimolar amounts of Na2S, reduced glutathione (both freshly neutralized), dithiothreitol, or dithioerythritol. In all cases, heating of the samples at 1000C for 5 min resulted in the loss of the 69K colored complex and most of the 21K and 26K protein bands. The data shown in Fig. 2C indicate that the high-molecular-weight colored material at the tops of gels of room-temperature-solubilized chromatophores was enriched in the 32K, 26K, and 21K proteins. To verify this, the top 3 mm of an analytical gel was cut out and rerun after various treatments. This material produced five bands; one of these corresponded to the outer membrane major protein, whereas the others consisted of the 32K, 26K, and 21K proteins plus a fourth protein with an apparent molecular weight of 48,000 (Fig. 3). Only a trace of the 26K protein remained after heating at 1000C, and the 21K protein was completely lost. Presence of similar modifiable proteins in the chromatophores of other photosynthetic bacteria. Since triads of proteins with molecular weights of between 19,000 and 32,000 have been reported for other photosynthetic bacteria (3, 25, 28, 39, 42), an investigation was undertaken to determine whether similar modifiable proteins are present in other photosynthetic bacteria. The patterns of bands for the four Chromatiaceae species tested were markedly similar. When the chromatophores of T. roseopersicina, T. violacea, and A. roseus were dissolved in sample buffer at room temperature and analyzed, all produced colored complexes (visible before staining) with apparent molecular weights, respectively, of 71,000, 74,000, and 70,000 (Fig. 3). However, the colored complex of T. roseopersicina was relatively faint. When the chromatophores of these organisms were heated at 1000C before electrophoresis, the colored complexes disappeared, and heavy-staining material appeared at the top of the gels (Fig. 4, B gels). In addition, two prominent bands disappeared or dimhed in intensity from the gels of T. roseopersicina, T. violacea, and A. roseus. These bands had apparent molecular weights of 26,000 and 21,000, 26,000 and 23,000, and 26,000

J. BACTERIOL.

1

2

k Om

3

.

32k 26k

oilp.-V01

FIG. 3. SDS-PAGE of chromatophore fr-actions. Room-temperature-solubilized chromatophores were subjected to electrophoresis9 in a 9% acrylamide gel. The pgmented area at the top of the gel was cut out and sliced into 1-cm lengths. These were placed in 1 ml of sample buffer and soaked for 15 min, followed by heating at 100)'C for 5 min (slot 1) or 500C for 10 min (slot 2). The strips uwere inserted in sample wells and -subjected to SDS-PAGE in a 10% gel. Slot 3 contains chromatophores heated to 50'C for 10 min. All abbreviations9 are as indicated for Fig. 1.

and 20,000, respectively (Fig. 4). When one-dimension strips of room-temperature-solubfiizd chromatophores of these three orgnsswere soaked and heated to 10000 and run in the second dimnension, part of the colored complexes of T. violacea and A. roseus and the three pairs of proteins affected by heat treatment remained in the first-dimnension strips (Fig. 4,0C gels). With A. roseus chromatophores, the 26K band was faint and diffuse (Fig. 4,30). The bands, labeled "OM," that appeared in each of the heated samples migrated in the same position as the major protein present in the Triton-insoluble fraction isolated from each of these species (Lane and Hurlbert, unpublished data). It is likely that this represents outer membrane contamination. It should be further noted that a protein with properties similar to the 25K protein of C. vinosum was present in two-dimension gels of each of the other Chromatiaceae chromatophores. A number of other differences in the banding pattern of room-temperature and 1000C-solubilized chromatophores were noted, but were not inves-

tigated. The pictures for the four Rhodospirillaceae species were generally similar. Room-tempera-

-

*-:-:o.:Nw

MODIFIABLE CHROMATOPHORE PROTEIN

VOL. 138, 1979 S

..

-ls

Jokik

...

CC

...

t^.: zcL

=L

s:

:.

,,.

=

.

-

.-

cc--

-I-C:

-cmcc

--4-CC

w >lyg

-OM

z

--|

:l

w-

213

s~-O -23K

-26K

,'.',iE*s.

-21 K

-26K -20K

-

w

^t

B

C

1

B

2

c

A

a

C

3

at 100°C. (1) T. roseopersicina; (2) T. violacea; (3) A. roseus. (A) Solubilized at room temperature and (B) at 100°C as described in the legend to Fig. 1. (C) Prepared by soaking a one-dimensional SDS-PAGE-run strip of room-temperature-solubilized chromatophore material in sample buffer for 15 min, followed by heating at 100°C for 5 min, after which it was run in second-dimension SDS-PAGE. The material which failed to migrate in the second dimension was detected by staining the first-dimension gel with Coomassie brilliant blue. All abbreviations are as for Fig. 1.

ture-solubilized chromatophores of Rp. capsulata and Rp. sphaeroides lacked any visible colored bands, whereas Rp. palustris Kl had a very faint colored band with a molecular weight of 70,000 and the 42 strain had a strong colored band with the same molecular weight (Fig. 5, 1A and 2A). All four species tested had at least two predominant proteins with apparent molecular weights of between 19,000 and 27,000 that became immobilized in the first-dimension gels upon heating (Fig. 5). However, the colored band in the two Rp. palustris strains did not become insoluble upon heating. In addition, Rp. palustris 42 contained a third predominant modifiable protein with a molecular weight of 42,000 (Fig. 5, 20), and both Rp. capsulata and Rp. sphaeroides contained several other proteins that became insoluble at 1000C (Fig. 5, 3C and 40). DISCUSSION The experiments reported in this paper show that the chromatophores of C. vinosum and six other photosynthetic bacteria contain a variety of proteins that can be modified by a number of environmental conditions. These conditions include the age of the sample, the temperature of solubilization, and the composition of the solubilization solution. W)hen freshly prepared chromatophores of C. vinosum were solubilized at low temperatures, a series of pigmented highmolecular-weight components was seen at the top of SDS-polyacrylamide gels; however, these bands were not seen when fresh samples were heated at temperatures of 500C or greater or

when samples were repeatedly frozen and thawed. The data suggest that these bands are incompletely solubilized complexes held together by easily disrupted bands. In addition to the high-molecular-weight pigmented bands, all of the Chromatiaceae species tested, as well as two strains of Rp. palustris, were found to contain a protein-pigment complex that was seen when chromatophores were solubilized at temperatures below 1000C. The nature of this complex is unknown, but the fact that it contains a number of low-molecular-weight components suggests that it may be part of the light-harvesting complex which has been shown in C. vinosum and other photosynthetic bacteria to contain proteins in the 7,000- to 12,000-molecularweight range (2, 23, 26). Of considerable interest was the observation that the chromatophores of C. vinosum and six other photosynthetic bacteria contain at least two proteins of similar molecular weights that became immobilized in the first-dimension gel when exposed to conditions which solubilize most other membrane proteins. The factors effecting these modifiable proteins are complex. For C. vinosum, heating alone was not sufficient to cause immobilization, since proteins in onedimension gels that were soaked and heated to 1000C in SDS alone did not become immobile, although some aggregation of the 21K protein did occur. Furthermore, the concentration of SDS appears to be important, since part of the proteins in gels which were heated in buffer alone (a procedure which diluted the SDS) did

214

J. BACTERIOL.

HUI AND HURLBERT

IMF -IS

_ l.s

cc-

cc-

_a-42K

-27K

- 22K .

B

A

-20K

c

A

1

8

2 -is

-is

-OM %:-.:.%:...'.

-

35.2 K

-OM *_-, 3K

-23K _22K

A

C

B

-

A

B4

21K

C

3 FIG. 5. SDS-PAGE gel of chromatophore of Rhodospirillaceae species solubilized at room temperature and at 100°C. (1) Rp. palustris Ki; (2) Rp. palustris 42; (3) Rp. capsulata; (4) Rp. sphaeroides. All conditions as described in the legend to Fig. 4. Abbreviations as for Fi. 1.

become immobile. Finally, fl-ME, as well as other sulfhydryl compounds, stimulated the process, as evidenced by the fact that the presence of fl-ME with SDS caused part of the 69K colored complex and most of the 26K and 21K

proteins to become immobile. The 21K protein was more sensitive to the modifying conditions since it aggregated at 600C, whereas the 26K protein was unaffected under these conditions. A plot of the log of the mobility of the 21K

VOL. 138, 1979

MODIFIABLE CHROMATOPHORE PROTEIN

aggregates from Fig. 2C versus the band number produced a straight line, which indicates that they represent a series of integral multiples of a basic monomeric unit (36). Also present in all of the Chromatiaceae chromatophores examined was a protein which had an apparent molecular weight of approximately 16,000 in unheated samples, but which was modified by heating alone to an apparent molecular weight of 25,000. Interpretation of the quantitative changes in the bands is difficult because of the large number of proteins present in chromatophores. A comparison of one-dimensional gels of heated and unheated material shows that protein bands are often still visible, although usually diminished in staining intensity, at the position of the modified proteins. Three explanations (or a combination) can account for this: (i) the proteins are incompletely modified so that only a part ofthe protein is lost, the rest migrating to the original position in the gel; (ii) two or more proteins comigrate, and only one of them is lost after a particular treatment; or (iii) a second protein is modified by the heating so that it migrates to the same position previously occupied by a protein which has in turn migrated to a new position. In the case of C. vinosum, all three effects applied to the 26K protein; i.e., it was not completely modified by heating (Fig. 2B and Fig. 3, lane 1), the 16K protein shifted to a similar molecular weight upon heating (Fig. 2B), and another protein comigrated with it in most gels (this protein could occasionally be resolved as a band running just ahead of the 26K band [Fig. 3, lane 3]). Recently Shepherd and Kaplan (37) have reported similar findings in a study made with chromatophores of Rp. sphaeroides. Our study confirms their observation regarding the effect of heating in SDS and ,8-ME on the 21K, 24K, and 35.2K proteins of Rp. sphaeroides and shows that the chromatophores of five other photosynthetic bacteria species contain at least two polypeptides in the 19- to 26-kdalton range that migrate differently after heating in SDSfl-ME solutions. However, certain of our findings and conclusions differ significantly from those of Shepherd and Kaplan (37). We found with C. vinosum chromatophores that similar results were obtained when other sulfhydryl compounds were substituted for the /3-ME. These data show that the observed changes in C. vinosum were a general consequence of the use of a reductant and not due to the specific action of fl-ME as was reported by Shepherd and Kaplan (37). In addition, Shepherd and Kaplan (37) have proposed that the Rp. sphaeroides proteins became insoluble as a result of aggregation with other chromatophore polypeptides. Such an explanation is not compatible with our results in

215

view of the manner in which our experiments were performed. Since our samples were first separated by one-dimensional gel electrophoresis, it is unlikely that peptides required for aggregation would have comigrated with each of the proteins which became modified upon subsequent heating. These data suggest that the immobilization of the chromatophore proteins does not require additional polypeptides and is of the self-aggregation type (33). In the case of the 21K protein of C. vinosum, this assumption was substantiated by the fact that it formed an aggregate series (Fig. 20). Several pieces of data indicate that the 21K and 26K proteins of C. vinosum are reaction center proteins. First, C. vinosum chromatophores contain three prominent proteins with molecular weights similar to those reported for the reaction center protein in C. vinosum as well as other photosynthetic bacteria (14, 23, 26, 28, 30, 39, 42). Secondly, Mechler and Oelze (23) found a similar triad in the isolated reaction center of C. vinosum and observed that the two lower-molecular-weight proteins were lost when the reaction center was heated at 700C. Finally, we fractionated the C. vinosum chromatophores by the procedure of Garcia et al. (13) and found that the "heavy fraction," which was reported to contain the reaction center, was enriched in the 32K, 26K, and 21K proteins, as well as in the 69K colored complex. When this material was heated to 1000C, the colored complex and the 26K and 21K proteins disappeared completely. Since it has been established that the two reaction center proteins of Rp. sphaeroides are modifiable (3, 37) and since the reaction centers of a number of photosynthetic bacteria, including Rs. rubrum (28, 42), Rp. sphaeroides (3, 30, 37, 39), Rp. capsulata (26), and C. vinosum (14,23), contain a triad of predominant proteins in the 19- to 30-kdalton range, it is possible that some reaction center proteins of most, and perhaps all, photosynthetic bacteria will behave similarly under the modifying conditions used in this study. It is reasonable to assume that the basic physical structure of proteins involved in the conversion of electromagnetic energy to chemical energy has been conserved throughout evolution; therefore, it follows that the reaction center proteins of all photosynthetic organisms will be similar in size and in response to conditions that affect the physical state of the functional regions of these molecules. However, verification of this hypothesis must await the isolation and analysis of reaction centers of all the organisms examined in this study, as well as from a large number of other photosynthetic species. It is not yet known whether non-reaction-cen-

216

HUI AND HURLBERT

ter proteins in photosynthetic as well as nonphotosynthetic systems respond to these effects in a similar fashion; however, there appear to be other proteins in some of the chromatophores, such as the 35.2K protein of Rp. sphaeroides, the 42K protein of Rp. palustris 42, and several proteins in Rp. capsulata (Fig. 50), that may not be reaction center or light-harvesting proteins, but which become insoluble upon heating in sample buffer. If this is the case, it is clear that use of,-ME (and other sulfhydryl reagents) in solubilization of proteins for SDS-PAGE analysis may lead to erroneous results and to difficulty in interpretation. On the other hand, it may be that all proteins which become insoluble upon exposure to the modifying conditions used here have a common unique structure which is required for certain membrane functions. Finally, these data show that extreme care must be taken in performing PAGE analysis of chromatophores and in interpreting the results of such analyses. It is clear that a number of chromatophore polypeptides have a narrow range of conditions (i.e., a "window") of solubilization outside of which they are either lost completely or quantitatively changed. A thorough PAGE study of chromatophores must, therefore, include an examination of these conditions on the protein pattern. LITERATURE CITED 1. Clayton, R. K. 1966. Spectroscopic analysis of bacteriochlorophyll in vitro and in vivo. Photochem. Photo-

biol. 5:669-677. 2. Clayton, R. K., and B. B. Clayton. 1972. Relations between pigments and proteins in the photosynthetic membranes of Rhodopseudomonas spheroides. Biochim. Biophys. Acta 283:492-504. 3. Clayton, R. K., and R. Haselkorn. 1972. Protein components of bacterial photosynthetic membranes. J. Mol. Biol. 68:97-105. 4. Clayton, R. K., and R. T. Wang. 1971. Photochemical reaction centers from Rhodopseudomonas spheroides. Methods Enzymol. 23:696-704. 5. Cohen-Bazire, G., and R. Kunisawa. 1960. Some observations on the synthesis and function of the photosynthetic apparatus in Rhodospirillum rubrum. Proc. Natl. Acad. Sci. U.S.A. 46:1543-1553. 6. Cohen-Bazire, G., and R. Kunisawa. 1963. The fine structure of Rhodospirillum rubrum. J. Cell Biol. 16: 401-418. 7. Collins, M. L P., and R. A. Niederman. 1976. Membranes of Rhodospirillum rubrum: physicochemical properties of chromatophore fractions isolated from osmotically and mechanically disrupted cells. J. Bacteriol. 126:1326-1338. 8. Drews, G. 1965. Die isolierung schwefelfreier purpurbakterien. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Suppl. 1:170-178. 9. Drews, G., R. Dierstein, and A. Schumacher. 1976. Genetic transfer of the capacity to forn bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata. FEBS Lett. 68:132-136. 10. Drews, G., and V. Witzemann. 1971. Zur taxonomie von Rhodopseudomonas palustris. Arch. Mikrobiol. 78:

J. BACTERIOL. 322-339. 11. Fraker, P. J., and S. Kaplan. 1971. Isolation and fractionation of the photosynthetic membranous organelles from Rhodopseudomonas spheroides. J. Bacteriol. 108:

466-473. 12. Fraker, P. J., and S. Kaplan. 1972. Isolation and characterization of a bacteriochlorophyll containing protein from Rhodopseudomonas spheroides. J. Biol. Chem. 247:2732-2737. 13. Garcia, A., L. P. Vernon, and H. Mollenhauer. 1966. Properties of Chromatium subchromatophores particles obtained by treatment with Triton X-100. Biochemistry 5:2399-2407. 14. Halsey, Y. D., and B. Byers. 1975. A large photoreactive particle from Chromatium vinosum chromatophores. Biochim. Biophys. Acta 387:349-367. 15. Holt, S. C., and A. G. Marr. 1965. Isolation and purification of the intracytoplasmic membrane of Rhodospirillum rubrum. J. Bacteriol. 89:1413-1420. 16. Huang, G. W., and S. Kaplan. 1973. Membrane proteins of Rhodopseudomonas spheroides. HII. Isolation, purification, and characterization of cell envelope proteins. Biochim. Biophys. Acta 307:301-316. 17. Hurlbert, R. E., J. R. Golecki, and G. Drews. 1974. Isolation and characterization of Chromatium vinosum membranes. Arch. Microbiol. 101:169-186. 18. Hurlbert, R. E., and J. Lascelles. 1963. Ribulose diphosphate carboxylase in Thiorhodaceae. J. Gen. Microbiol. 33:445458. 19. Ketchum, P. A., and S. C. Holt. 1970. Isolation and characterization of the membranes from Rhodospirillum rubrum. Biochim. Biophys. Acta 196:141-161. 20. King, M. T., and G. Drews. 1973. The function and localization of ubiquinone in the NADH and succinate ozidase systems of Rhodopseudomonas palustris. Biochim. Biophys. Acta 305:230-248. 21. King, J., and U. K. Laemmli. 1971. Polypeptides of the tail fibres of bacteriophage T4. J. Mol. Biol. 62:465477. 22. Lien, S., H. Gest, and A. San Pietro. 1973. Regulation of chlorophyll synthesis in photosynthetic bacteria. J. Bioenerg. 4:423-434. 23. Mechler, B., and J. Oelze. 1978. Differentiation of the photosynthetics apparatus of Chromatium vinosum, strain D. II. Structural and functional differences. Arch. Microbiol. 118:99-108. 24. Niederman, R. A., B. J. Segen, and K. D. Gibson. 1972. Membranes of Rhodopseudomonas spheroides. I. Isolation and characterization of membrane fractions from extracts of aerobically and anaerobically grown cells. Arch. Biochem. Biophys. 152:547-560. 25. Nieth, K. F., and G. Drews. 1974. The protein patterns of intracytoplasmic membranes and reaction center particles isolated from Rhodopseudomonas capsulata. Arch. Microbiol. 96:161-174. 26. Nieth, K. F., and G. Drews. 1975. Formation of reaction center and light harvesting bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata. Arch. Microbiol. 104:77-82. 27. Nieth, K. F., G. Drews, and R. Feick. 1975. Photochemical reaction centers from Rhodopseudomonas capsulata. Arch. Microbiol. 105:43-45. 28. Noel, H., M. V. D. Rest, and G. Gingras. 1972. Isolation and partial characterization of a P870 reaction center complex from wild type Rhodospirillum rubrum. Biochim. Biophys. Acta 275:219-230. 29. Oelze, J., and G. Drews. 1972. Membranes of photosynthetic bacteria. Biochim. Biophys. Acta 265:209-239. 30. Okamura, M. Y., L A. Steiner, and G. Feher. 1974. Characterization of reaction centers from photosynthetic bacteria. I. Subunit structure of the protein mediating the primary photochemistry in Rhodopseudomonas sphaeroides R-26. Biochemistry 13:1394-1410.

VOL. 138, 1979

MODIFIABLE CHROMATOPHORE PROTEIN

31. Prince, R. C., and A. R. Crofts. 1973. Photochemical reaction centers from Rhodopseudomonas capsulata ala pho+. FEBS Lett. 35:213-216. 32. Reed, D. W., D. Raveed, and H. W. Israel. 1970. Functional bacteriochlorophyll-protein complexes from chromatophores of Rhodopseudomonas spheroides strain R-26. Biochim. Biophys. Acta 223:281-291. 33. Reitheima, R. A. F., and P. D. Bragg. 1977. Molecular characterization of a heat-modifiable protein from the outer membrane of Escherichia coli. Arch. Biochem. Biophys. 178:527-534. 34. Russell, R. R. B. 1975. Two-dimensional SDS-polyacrylamide gel electrophoresis of heat-modifiable outermembrane proteins. Can. J. Microbiol. 22:83-91. 35. Schnaitman, C. A. 1971. Solubilization of the cytoplasmic membrane of Escherichia coli by Triton X-100. J. Bacteriol. 108:545-552. 36. Scurzi, W., and W. N. Fishbein. 1973. The geometric mobility sequence in polyacrylamide gel electrophoresis of polymeric proteins: its significance for polymer geometry and electrophoretic theory. Trans. N.Y. Acad. Sci. 35:396-416. 37. Shepherd, W. D., and S. Kaplan. 1978. Effect of heat and 2-mercaptoethanol on intracytoplasmic membrane polypeptides of Rhodopseudomonas sphaeroides. J. Bacteriol. 135:656-667. 38. Stanier, R. Y. 1963. The organization of the photosyn-

39.

40.

41.

42.

43.

217

thetic apparatus in purple bacteria, p. 242-252. In D. Mazia and A. Tyler (ed.), General physiology of cell specialization. McGraw-Hill, Inc., New York. Steiner, L A., My. Y. Okamura, A. D. Lopes, E. Moskowitz, and G. Feher. 1974. Characterization of reaction centers from photosynthetic bacteria. II. Amino acid composition of the reaction center protein and its subunits in Rhodopseudomonas sphaeroides R26. Biochemistry 13:1403-1410. Takemoto, J., and J. La_lles. 1974. Function of membrane proteins coupled to bacteriochlorophyll synthesis. Studies with wild type and mutant strains of Rhodopseudomonas sphaeroides. Arch. Biochem. Biophys. 163:507-514. Tonn, S. J., G. E. Gogel, and P. A. Loach. 1977. Isolation and characterization of an organic solvent soluble polypeptide component from photoreceptor complexes of Rhodospirillum rubrum. Biochemistry 16:877-885. Wang, R. T., and R. K. Clayton. 1973. Isolation of photochemical reaction centers from a carotenoidless mutant of Rhodospirillum rubrum. Photochem. Photobiol. 17:57-61. Worden, P. B., and W. R. Sistrom. 1964. The preparation and properties of bacterial chromatophore fractions. J. Cell Biol. 23:135-150.