A highdefinition native polyacrylamide gel ... - Wiley Online Library

The Plant Journal (2011) 67, 181–194

doi: 10.1111/j.1365-313X.2011.04577.x

TECHNICAL ADVANCE

A high-definition native polyacrylamide gel electrophoresis system for the analysis of membrane complexes Roman Ladig1, Maik S. Sommer3,6, Alexander Hahn3,6, Matthias S. Leisegang2,6, Dimitrios G. Papasotiriou4, Mohamed Ibrahim3,6, Rajae Elkehal6, Michael Karas4, Volker Zickermann5, Michael Gutensohn1,†, Ulrich Brandt3,5,6, Ralf Bernd Klo¨sgen1 and Enrico Schleiff2,3,6,* 1 Institute of Biology – Plant Physiology, Martin Luther University Halle-Wittenberg, Weinbergweg 10, D-06120 Halle/Saale, Germany, 2 Cluster of Excellence ‘Macromolecular Complexes’, Goethe University, Frankfurt am Main, Germany, 3 Center of Membrane Proteomics, Goethe University, Frankfurt am Main, Germany, 4 Institute of Pharmaceutical Chemistry, Goethe University, Max-von Laue-Str. 9, D-60438, Frankfurt am Main, Germany, 5 Molecular Bioenergetics, Medical School, Goethe University, Theodor-Stern-Kai 7, D-60590, Frankfurt am Main, Germany, and 6 Institute of Molecular Cell Biology of Plants, Goethe University, Max-von Laue-Str. 9, D-60438, Frankfurt am Main, Germany Received 16 December 2010; revised 7 March 2011; accepted 7 March 2011; published online 28 April 2011. * For correspondence (fax +49 (0) 69 798 29286; e-mail [email protected]). † Present address: Department of Horticulture and Landscape Architecture, Purdue University, 625 Agriculture Mall Drive, West Lafayette, IN 47907, USA.

SUMMARY Native polyacrylamide gel electrophoresis (PAGE) is an important technique for the analysis of membrane protein complexes. A major breakthrough was the development of blue native (BN-) and high resolution clear native (hrCN-) PAGE techniques. Although these techniques are very powerful, they could not be applied to all systems with the same resolution. We have developed an alternative protocol for the analysis of membrane protein complexes of plant chloroplasts and cyanobacteria, which we termed histidine- and deoxycholatebased native (HDN-) PAGE. We compared the capacity of HDN-, BN- and hrCN-PAGE to resolve the well-studied respiratory chain complexes in mitochondria of bovine heart muscle and Yarrowia lipolytica, as well as thylakoid localized complexes of Medicago sativa, Pisum sativum and Anabaena sp. PCC7120. Moreover, we determined the assembly/composition of the Anabaena sp. PCC7120 thylakoids and envelope membranes by HDN-PAGE. The analysis of isolated chloroplast envelope complexes by HDN-PAGE permitted us to resolve complexes such as the translocon of the outer envelope migrating at approximately 700 kDa or of the inner envelope of about 230 and 400 kDa with high resolution. By immunodecoration and mass spectrometry of these complexes we present new insights into the assembly/composition of these translocation machineries. The HDN-PAGE technique thus provides an important tool for future analyses of membrane complexes such as protein translocons. Keywords: native polyacrylamide gel electrophoresis, cyanobacteria, chloroplasts, thylakoid complexes, outer/inner envelope complexes, protein translocation.

INTRODUCTION Macromolecular membrane protein complexes play an important role in many cellular processes and thus their investigation is of importance for the understanding of cellular systems (see amongst others Amunts and Nelson, 2009; Tree et al., 2009; Schleiff and Tampe´, 2009; Zickermann et al., 2009; Groves and Kuriyan, 2010; Hurley and Hanson, 2010; Schmidt et al., 2010). Their identification as ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd

well as the analysis of their composition, subunit stoichiometry and assembly is still a challenging task. A number of methods have been established so far, mostly optimized for the particular system under study (Bulseco and Wolf, 2003; Slotboom et al., 2008; Crociani et al., Crociani, 2010; Hoffmann et al., 2010). Polyacrylamide gel electrophoresis (PAGE) technology is an important technique for investi181

182 Roman Ladig et al. gating many aspects of membrane complex function. The invention of the denaturing SDS-PAGE technique (Maizel, 1966) marked a revolution in protein analysis as every cell biological, biochemical or biophysical study includes this method at least at one point. Based on the success of denaturing PAGE, native PAGE systems have been explored and developed. One of the most remarkable developments is the so-called blue native (BN-) PAGE and its further development clear native (CN-) PAGE (Scha¨gger and von Jagow, 1991; Scha¨gger et al., 1994). These methods use non-ionic detergents with or without the addition of Coomassie in combination with the near neutral buffer capacity of Bis-TRIS (bis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane). These techniques were further optimized (Wittig et al., 2006; Reisinger and Eichacker, 2007; Wittig and Scha¨gger, 2008) and have helped to develop our understanding of many different membrane protein complexes. Blue native PAGE has been an indispensable tool for the analysis of the assembly and function of the mitochondrial and chloroplast electron transport chains (Scha¨gger and von Jagow, 1991; Jansch et al., 1995; Karpova and Newton, 1999; Millar et al., 2003). Amongst many other important observations BN-PAGE led to the discovery that proteins responsible for cytoplasmic male sterility are subunits of the mitochondrial complex V (Heazlewood et al., 2003; Sabar et al., 2003). Our understanding of the function and complex formation of the photosynthetic protein complexes and the plastidic F0F1-ATP synthase was similarly influenced by this technique (e.g. Huang et al., 1994; Seelert et al., 2003; Zhang et al., 2001; Heinemeyer et al., 2004; Rexroth et al., 2004; Komenda et al., 2004; Aro et al., 2005; Pribil et al., 2010). However, not only electron transport chains and photosynthetic complexes were analyzed by BN-PAGE. This system has also been employed for the characterization of the plastid RNA-polymerase complex (e.g. Suzuki et al., 2004), protein complexes of the plasma membrane of spinach (Kjell et al., 2004), microsomal membrane proteins from Arabidopsis (Drykova et al., 2003), complexes of the cyanobacterial cell wall (Moslavac et al., 2005) and many others. Another field where BN-PAGE technology has played an important role is the analysis of protein translocation into endosymbiotically derived organelles. For example, many important discoveries in mitochondrial protein import and its molecular mechanisms in general (e.g. Rehling et al., 2004) as well as in plant mitochondria in particular (e.g. Jansch et al., 1998; Werhahn et al., 2001, 2003) are based on this method. Similarly, BN-PAGE was applied to analyze the protein translocation into thylakoids (e.g. Berghofer and Klo¨sgen, 1999; Cline and Mori, 2001; Klostermann et al., 2002; Martin et al., 2009; Ma and Cline, 2010) and the envelope membranes of chloroplasts. For example, the two components Toc55 and Tic62 (Caliebe et al., 1997; Ku¨chler et al., 2002) and a 1 MDa TIC (translocon in the inner

envelope membrane of chloroplasts complex) were discovered by BN-PAGE (Kikuchi et al., 2009). Similarly, outer envelope import complexes were analyzed using this technique (Kikuchi et al., 2006; Chen and Li, 2007; Qbadou et al., 2007). However, not all membrane protein complexes could be successfully analyzed by the currently established BN-PAGE protocols, including complexes of the outer and inner envelope membranes which so far can only be analysed in the context of intact chloroplasts. We therefore developed a modified protocol termed histidine- and deoxycholatebased native (HDN-) PAGE with the capacity for a highresolution separation of membrane protein complexes of isolated chloroplast envelope membranes and cyanobacterial cell walls. At the same time we document that native PAGE techniques have to be specifically optimized for each biological system. RESULTS AND DISCUSSION Analysis of mitochondrial complexes by native PAGE To separate membrane protein complexes in chloroplast envelope membranes we have developed a new native PAGE system by combining key features of two different native gel systems (Table 1; see below). The recently invented high-resolution clear native (hrCN-) PAGE system employs a new charge-conferring detergent mixture, namely deoxycholic acid (DOC) and dodecyl maltoside (DDM; Table 1). Since Coomassie may compromise the native state depending on the complexity and origin of the analyzed sample, the DOC/DDM alternative has proved to be promising for samples that could previously not be processed by BN-PAGE. By combining this approach with a discontinuous native gel system (Niepmann and Zheng, 2006) we were able to overcome the limited resolution capability of the hrCN system by a native PAGE system engaging histidine and deoxycholate to separate native protein complexes. We termed this new procedure HDN-PAGE. To evaluate the capacity of the new gel system we compared the resolution of mitochondrial oxidative phosphorylation (OXPHOS) complexes. Following established protocols (Scha¨gger and Pfeiffer, 2000; Van Coster et al., 2001; Wittig and Scha¨gger, 2008) we were able to separate the respiratory chain supercomplexes (Figure 1a, S), the NADH-ubiquinone:oxidoreductase complex I (I), the ATPsynthase complex (V), the cytochrome-reductase complex (III) and the cytochrome-oxidase complex (IV) from bovine heart mitochondria (BHM) by BN-PAGE and hrCN-PAGE. The same sample was investigated by the new HDN-PAGE system, which did not perform better or worse than the established tools. The separation of the single respiratory chain complexes is sharper than with BN and hrCN, especially in the lower molecular weight range. However, by

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 67, 181–194

HDN-PAGE for membrane protein analysis 183 Table 1 The ingredients of the buffer systems used in this study. Major differences are highlighted in bold

BN-PAGE Sample buffer 50 mM Bis-TRIS pH 7.0 0.5 M ACA 10% glycerol 5 mM DTT 0.1 U/ll micrococcal nuclease 2 mM CaCl2 1 mM AEBSF 10 lM E64 favored detergent 8 g/g protein CBB G-250 Cathode buffer 50 mM Tricine 15 mM Bis-TRIS 0.02% CBB G-250 Gel buffer 50 mM Bis-TRIS pH 7.0 0.5 M ACA 10–20% glycerol Anode buffer 50 mM Bis-TRIS pH 7.0

hrCN-PAGE

HDN-PAGE

50 mM Bis-TRIS pH 7.0 0.5 M ACA 10% glycerol 5 mM DTT 0.1 U/ll micrococcal nuclease 2 mM CaCl2 1 mM AEBSF 10 lM E64 favored detergent

100 mM TRIS pH 8.0 0.5 M ACA 10% glycerol 5 mM DTT 0.1 U/ll micrococcal nuclease 2 mM CaCl2 1 mM AEBSF 10 lM E64 favored detergent

50 mM Tricine 15 mM Bis-TRIS 0.05% DOC 0.01% DDM

100 mM histidine-base 3 mM TRIS-base 0.05% DOC 0.01% DDM

50 mM Bis-TRIS pH 7.0 0.5 M ACA 10–20% glycerol

200 mM TRIS pH 8.8 – 10–20% glycerol

50 mM Bis-TRIS pH 7.0

100 mM TRIS pH 8.8

Abbreviations: BN-PAGE, blue native PAGE; hrCN-PAGE, high-resolution clear native PAGE HDNPAGE, histidine and deoxycholate based native PAGE; TRIS, 2-amino-2-(hydroxymethyl)-1,3propandiol; Bis-TRIS, bis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane; ACA, 2-aminohexanoicacid; DTT, dithiothreitol; AEBSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride; E64, N-(trans-epoxysuccinyl)-L-leucine 4-guanidinobutylamide; CBB, Coomassie Brilliant Blue; DDM, dodecyl maltoside; DOC, deoxycholic acid.

HDN-PAGE we did not preserve as many supercomplexes as with BN- and hrCN-PAGE. Analyzing protein complexes in the mitochondrial membranes of Yarrowia lipolytica (Figure 1b, lane 1, 3), we obtained the best separation using BN-PAGE. With hrCN-PAGE and HDN-PAGE the bands assigned to complexes III and IV were hardly visible. Furthermore, with HDN-PAGE we could not observe the high molecular weight band assigned to respiratory chain supercomplexes (S; Nu¨bel et al., 2009) and complex V dimer (V2). In a mutant that is known to destabilize complex I we detected a subcomplex of complex I with a molecular weight of approximately 650 kDa (Ix). Complete complex I and a subcomplex of the same size was found in wild-type mitochondria when analyzed by HDN-PAGE. We conclude that for Y. lipolytica and bovine heart mitochondria HDN-PAGE does not perform superior to the established BN-PAGE or hrCN-PAGE. Moreover, HDN-PAGE partly destabilized OXPHOS complexes of Y. lipolytica as monitored by decomposition of complex I of respiratory supercomplexes and of the dimeric form of complex V. Analysis of complexes in thylakoids of plants by native PAGE To further evaluate our system we have analyzed the separation of chloroplast and thylakoid complexes by the three

distinct native page systems (Figure 2), because native PAGE analysis is frequently used to analyze the complex composition of these membranes from cyanobacteria, algae and plants (e.g. Ossenbu¨hl et al., 2004; Danielsson et al., 2006; Schwenkert et al., 2006). At this stage we used two different plant sources, namely Medicago sativa and Pisum sativum, as well as the cyanobacterium Anabaena sp. PCC 7120. We observed a significant difference in resolution of the complexes with any of the native PAGE systems depending on the detergent used for solubilization. Specifically, DDM performed better than digitonin. In our hands, the resolution of the complexes by hrCN-PAGE was not satisfactory. The resolution quality observed using BNPAGE was (especially in the high molecular weight regions) better than by using HDN-PAGE, even though the migration pattern was slightly different (Figure 2a,b). Comparing the patterns with previously established annotations (e.g. Thidholm et al., 2002) we were able to tentatively assign photosystem II (PSII) supercomplexes, photosystem I (PSI), the light-harvesting complex and Rubisco. Remarkably, we observed differences in the migration behavior between the Rubisco complexes from P. sativum and M. sativa (Figure 2a,b; R, R1, R2). In BN-BAGE of digitonin-solubilized chloroplast samples one Rubisco complex migrated at 480 kDa for both chloroplast sources. Using DDM for


184 Roman Ladig et al. Figure 1. Native PAGE analysis of mitochondrial membranes. Solubilized mitochondria were subjected to histidineand deoxycholate-based native (HDN-) (left), blue native (BN-) (middle) or high-resolution clear native (hrCN-) PAGE (right) using the buffer systems listed in Table 1. (a) Bovine heart mitochondria, digitonin concentrations for solubilization as indicated [Dig (%)]. (b) Mitochondria isolated from wild-type Yarrowia lipolytica (wt, lanes 1, 3) and the DNB8M mutant strain (mt, lanes 2, 4), 1.5% digitonin throughout. Coomassie stain is shown in (a) lanes 1–3 and in (b) lanes 1 and 2; the corresponding NADH: nitrotetrazolium blue (NTB) activity stain (NADH) is shown in (a) lanes 4 (2% digitonin) and in (b) lanes 3 and 4; the bands corresponding to respiratory chain complexes are labeled I, III and IV, respectively. Y marks putative glycerol-3-phosphate-dehydrogenase and Ix marks a complex I subcomplex. Complex V was detected either in monomeric (V) or dimeric form (V2). Supercomplexes are labeled with S, which possibly contain complex I and complex V, which has to be further investigated. The molecular weight standard is indicated on the right side of each panel.

solubilization, an additional Rubisco complex migrating at 750 kDa was observed, but only in the case of chloroplasts from P. sativum. Comparison between the gel systems revealed even greater variations. No matter which detergent was used to solubilize P. sativum chloroplasts only one Rubisco complex at 480 kDa was observed in hrCN-PAGE while it migrated at 680 kDa in HDN-PAGE. Different explanations might be given for this behavior. On the one hand, binding of chargeconferring compounds may vary markedly between different proteins and thus may have different effects on the migration pattern of different protein complexes. However, this explanation is somewhat compromised by the observed migration behavior of the Rubisco complex from M. sativa migrating at 480 kDa irrespective of the gel system or detergent used. On the other hand, it is known that Rubisco complexes can interact with each other or with the chloroplastic cpn60 system (e.g. Ellis, 1987; Irving and Robinson, 2006). Such interaction might be sensitive to detergent and charged compounds added during solubilization and separation. Whether the observed shift of the Rubisco complex

to higher molecular masses under some conditions of native PAGE indeed reflects such higher assembly intermediates will require further investigation. Like with respiratory chain complexes, higher-order complexes that most likely represented multimers of PSII were observed with BN-PAGE and with HDN-PAGE (Figure 2c). While analyzing protein complexes with the BN-PAGE system we observed a precipitation of protein in the higher molecular weight complexes, a phenomenon not observed with HDN-PAGE (Figure 2c). Thus, for the analysis of the PSII supercomplexes HDNPAGE might be an efficient tool alone and in addition to BN-PAGE. Complexes in thylakoids and the cell wall of cyanobacteria We next inspected the composition of the thylakoid membrane of the cyanobacterium Anabaena sp. PCC 7120 (Figure 3). Here we observed a comparable resolution with HDN-PAGE when compared with BN-PAGE. In total we observed 13 complexes in the range of 70 kDa to 1 MDa as estimated from the molecular mass standards. To confirm the assignment of the complexes based on the analysis of


HDN-PAGE for membrane protein analysis 185 the sample solubilized with DDM. The band assigned to photosystem II supercomplexes (IIS), of photosystem I (I), of photosystem I and II subcomplexes (IS, IIS), of the light-harvesting complexes (L) and of Rubisco (R) according to (Thidholm et al., 2002) are marked. NADH: NBT oxidoreductase activity (*) and the presence of chlorophyll (+) are indicated. The molecular masses of the standard are indicated on the left side of each panel. (c) The high molecular mass region of the thylakoid membrane complexes of Medicago sativa solubilized by dodecyl maltoside and separated by HDN(left) and BN-PAGE (right) as in (a) is shown. Triangles indicate the larger complexes observed only in BN-PAGE. The molecular masses of the standard used are as indicated.

Figure 2. Separation of complexes localized in plant thylakoids by native PAGE. Chloroplast from Medicago sativa (a) and Pisum sativum (b) equivalent to 15 lg of chlorophyll were solubilized in 30 ll sample buffer containing either 1.5% digitonin (Dig, lane 1) or 1% dodecyl maltoside (DDM, lanes 2 and 3) and subjected to the three native gel systems (histidine- and deoxycholate-based native, HDN, left; blue native, BN, middle; high-resolution clear native, hrCN, right). Lanes 1 and 2 show the Coomassie-stained gel and lane 3 the in-gel NADH:nitrotetrazolium blue (NBT) oxidoreductase activity staining (NADH) of

Nostoc punctiforme (Cardona et al., 2009) we analyzed the proteins of the complexes indicated (Figure 3) by mass spectrometry after in-gel trypsin digestion. We could confirm that the six complexes migrating between 900 and 390 kDa contained subunits of PSI (Table 2). In the complexes migrating at 800 and 400 kDa we found PSI components only, suggesting that these are the monomeric and dimeric forms of the photosystem. In the remaining complexes in this mass range in addition to PSI components we identified subunits of phycobilisomes (complexes 2 and 3 at 700 and 620 kDa), of ATP synthase (complex 4 at 590 kDa) or of PSII (complex 5 at 430 kDa). We also verified the migration of PSII at 350 kDa (complex 7) and of the cytochrome-b6/f complex at 280 kDa (complex 8) as suggested by Cardona et al. (2009). Moreover, we identified an additional dominant complex at 240 kDa containing PSII proteins (complex 9). Migrating at molecular weights below 200 kDa we observed, for example, SecD (160 kDa), phycocyanin (130 and 110 kDa) and protochlorophyllide oxidoreductase (110 kDa). Even though we did not analyze individual protein spots in a second dimension gel this confirmed that we are able to detect the different supercomplexes by HDN-PAGE. In addition to these known components we identified five proteins of unknown function (Table 2; Alr2489, All3941, Alr0199, Alr3790, Alr4119). However, because we also found contamination by very abundant outer membrane proteins (Alr4550, All4499, Alr0834, All3041) we could not decide with certainty whether the unknown proteins were indeed components of thylakoidal complexes. In addition to thylakoid membranes we explored the separation of complexes in the cell wall fraction of Anabaena sp. PCC 7120. The first attempts to separate these complexes by BN-PAGE had not been very successful (Moslavac et al., 2005). After solubilization with either 1.5% (w/v) digitonin or 1% (w/v) DDM, we observed that solubilization by digitonin resulted in better resolution but that DDM resulted in better solubilization of the complexes (Figure 3b). In addition, we noted that for this membrane resolution with hrCN-PAGE was somewhat superior to that with BN-PAGE (lane 2). However, in this case by far the best resolution was obtained with HDN-PAGE. Thirteen well-defined bands in the range of 90 kDa up to 1 MDa could be perceived.


186 Roman Ladig et al.

Figure 3. Separation of complexes localized in cyanobacterial membranes by native PAGE. Thylakoids of Anabaena sp. PCC 7170 equivalent to 15 lg of chlorophyll (a) or cell wall fractions with 20 lg protein (b) were solubilized in 30 ll sample buffer containing either 1.5% digitonin (Dig, lane 1) or 1% dodecyl maltoside (DDM, lanes 2 and 3) and subjected to the three native gel systems (histidine- and deoxycholate-based native, HDN, left; blue native, BN, middle; high-resolution clear native, hrCN, right). Lanes 1 and 2 show the Coomassiestained gel and lane 3 the in-gel NADH:nitrotetrazolium blue (NBT) oxidoreductase activity staining (NADH) of the sample solubilized with DDM. NADH: NBT oxidoreductase activity (*) and the presence of chlorophyll (+) are indicated. The assignment of the monomeric (I) or dimeric (I2) state of photosystem I, of photosystem II (II) and of the cytochrome-b6/f complex are indicated according to (Cordona et al., 2009). The molecular masses of the standard used are indicated on the left of each panel. Arrowheads point to analyzed bands (Tables 2 and 3).

We next used mass spectrometry to analyze the protein content in the individual complexes (Table 3). We made the observation that the band migrating at approximately 430 kDa (complex 5) was contaminated with the identified PSI and PSII complex. Furthermore, we noticed that all fractions analyzed in the low molecular weight region of the gel (up to 370 kDa) contained the protein Alr1819, which was annotated as a cell surface protein (Moslavac et al., 2005;

Nicolaisen et al., 2009a,b). Thus, it appears likely that only the complexes at 140 and 160 kDa are specific for this protein. Similarly, all analyzed bands migrating with a molecular weight larger than 420 kDa contain the two porins Alr4550 and All4499, which were previously noticed to be the most abundant porin-like proteins of the outer membrane of Anabaena sp. PCC 7120 (Moslavac et al., 2005, 2007a,b). As porins typically form trimeric complexes it has to be assumed that these proteins do not migrate properly. Despite this, we identified three proteins not noticed previously in the outer membrane fraction: The first is the protein encoded by all7614 with similarity to the porin OprB in the complex of about 500 kDa. The second protein is a nitratebinding protein with a predicted TAT signal sequence encoded by alr0608 migrating at approximately 90 kDa and thus possibly reflecting a dimer. The third protein is encoded by alr2489. The protein is a putative plasma membrane protein and might represent a contamination. Remarkably, however, the protein was not found as contamination in previous attempts to analyze the outer membrane proteome (Moslavac et al., 2005, 2007a,b). We further identified the most prominent Omp85 (Ertel et al., 2005; Nicolaisen et al., 2009b) encoded by alr2269 in multiple but distinct complexes. In a previous attempt to analyze complexes of the outer membrane by BN-PAGE this protein was identified on native PAGE migrating at approximately 370 and 250 kDa (Moslavac et al., 2005), which is consistent with the tetrameric composition determined for the isolated and reconstituted protein by cross-linking (Bredemeier et al., 2007). Here we find the protein migrating at 180, 250 and 370 kDa, which could reflect the homodimeric, trimeric and tetrameric state of the protein. In addition, we observed a significant portion of the protein at 230 and 450 kDa. In contrast to the previous analysis using BN-PAGE (Moslavac et al., 2005), the TolC homologue of Anabaena sp. PCC 7120 (Moslavac et al., 2007b) was migrating at higher molecular weights, namely at 450 and 600 kDa based on the molecular weight standard migration. Although in the first attempt presented here not all outer membrane proteins previously identified could be detected, the resolution is significantly better than that found while using BN-PAGE (Moslavac et al., 2005). Thus, this method appears to be particularly useful for investigating the complexes formed by Omp85, TolC and of porins. Separation of distinct outer envelope translocon complexes by native PAGE Up to now, chloroplast envelope complexes have been analyzed in the context of intact chloroplasts only, because resolution of these membranes in BN-PAGE was not satisfactory (Figure 4). Thus, one aim of developing HDNPAGE has been to obtain better resolution of protein complexes from chloroplast envelopes. Comparing the


HDN-PAGE for membrane protein analysis 187 Table 2 Mass spectrometric analysis of Anabaena sp. PCC 7120 thylakoid membrane complexes Spot MWa 1

2

3

4

5

6

7

8

9

10 11

12

13

Name

800(I3) psaA psaB psaC psaD psaF 700 psaA psaB apcE 620 psaA psaB apcE 590 (I2) psaA psaB atpB 430 psaA psaB psaD psaF psbA (D1) psbD (D2) 390 (I) psaA psaB psaC psaD psaF 350 (II) psbA (D1) psbA (D1) psbB (CP47) psbC (CP43) psbD (D2) psbH 280 petA petB petC petD 240 psbA (D1) psbA (D1) psbB (CP47) psbD (D2) ? 160 secD 130 cpcA cpcB ? 110 psbC (CP43) psbO cpcA cpcB por ? 70 atpB cpcA psbO ? ?

Gene

MWPb MSCc Complexd

alr5154 84 alr5155 84 asr3463 9 all0329 15 all0109 18 alr5154 84 alr5155 84 alr0020 127 alr5154 84 alr5155 84 alr0020 127 alr5154 84 alr5155 84 all5039 52 alr5154 84 alr5155 84 all0329 15 all0109 18 all3572 40 alr4290 40 alr5154 84 alr5155 84 asr3463 9 all0329 15 all0109 18 alr4866 40 all3572 40 all0138 56 alr4291 50 alr4290 39 asl0846 7 all2452 36 alr3421 24 all2453 19 alr3422 31 all4866 40 all3572 40 all0138 56 alr4290 39 alr2489 22 all0121 51 alr0529 18 alr0528 19 all3941 36 alr4291 50 all3854 30 alr0529 18 alr0528 19 all1743 37 alr0199f 20 all5039 52 alr0529 18 all3854 30 alr3790 17 alr4119 16

269 319 174 58 148 114 169 131 218 185 139 177 151 132 230 161 137 46 90 64 257 186 136 278 127 363 363 622 367 107 65 155 284 282 277 399 155 688 110 92 154 767 212 415 613 96 250 82 86 468 1380 148 1230 138 138

2 · PSI

PSI and PB

PSI and PB

a

Estimated molecular weight in kDa. Molecular weight predicted. c Mascot score. d Contamination by outer membrane proteins were observed (All3051, Alr4550, All4499, Alr0834), which are not inserted into the table. e Contamination of PSII components was detected due to the close migration of bands 8 and 9. f Similar to allophycocyanin alpha subunit from Microcystis aeruginosa NIES-843. Abbreviations for the complexes are: ATP, ATP synthase; Cyt, Cyt-b6/f complex; PB, phycobilisomes; PC, phycocyanin; por, protochlorophyllide oxido-reductase; PSI, photosystem I; PSII, photosystem II; T, protein translocon. b

PSI and ATP

PSI and PSII

PSI

PSII

Cyte

PSII

T PC

PC

?

The identified proteins correspond to the marked bands (white arrowheads) found by histidine- and deoxycholate-based native (HDN-) PAGE analyses (Figure 3a) from top to bottom.

three different native PAGE systems applied to these membranes we were able to resolve distinct Coomassie stainable complexes only with HDN-PAGE (Figure 4a,b), particularly when using 2% (w/v) digitonin as the solubilizing detergent (lane 6). In the outer envelope vesicles we observed several complexes with an apparent molecular mass between 100 and 750 kDa when solubilizing the membrane with digitonin (Figure 4a; Table 4). Several of these complexes could also be observed while analyzing membranes solubilized with 2% (w/v) DDM. Four of these are assemblies with TOC (translocon in the outer envelope membrane of chloroplasts) components, because the complexes with 130, 250, 420 and 700 kDa were detected with Toc34 antibodies (Figure 4a, left, lane 7). Three of those complexes with 130, 420 and 700 kDa were also observed as very diffuse bands in a western blot after hrCN-PAGE (Figure 4a, right, lane 7), whereas with BN-PAGE only a very diffuse band at higher molecular weight was detectable. Remarkably, the 130 and 420 kDa complexes had also been observed in entire chloroplast membranes (Kikuchi et al., 2006) and found to contain Toc75 and Toc34 only. However, the major complex found in these studies migrates at an apparent molecular mass of ‡800 kDa (Kikuchi et al., 2006; Chen and Li, 2007). According to Kikuchi et al. (2006) a large portion of Toc159 still contains the acidic A-domain that is absent in isolated envelope vesicles. This additional domain and a possible abnormal migration caused by its acidic nature could explain the size difference observed between these studies and our findings with isolated outer envelope vesicles. To support this notion we analyzed some of the bands observed while separating outer envelope complexes by mass spectrometry (Figure 4a, left; Table 4). The 700, 500 and the 250 kDa bands display all the TOC core complex components, namely Toc34, Toc159 and Toc75. The composition of the 500 kDa band, however, was somewhat unexpected, because Toc34 antibodies did not detect the 500 kDa band (Figure 4a, left). Unfortunately, after several attempts we were unable to identify proteins of the band migrating at 420 kDa. In the complexes at 220 and


188 Roman Ladig et al. Table 3 Mass spectrometric analysis of Anabaena sp. cell wall complexes

Spot

MWa

1

1000

2

600

3

500

4

450

Name

oprB-I

hgdD

oprB-II hgdD omp85

Gene

MWPb

MSCc

alr4550 all4499 alr0834 alr4550 all4499 alr2887 alr4550 all4499 all7614 alr2887 alr4550 alr2269

61 59 54 61 59 81 61 59 58 81 61 90

1790 581 166 1830 592 482 1190 586 141 492 251 204

5

420d

alr2489 all3984

21 25

70 135

6

400

alr4550 all3984

61 25

129 531

7

370

all4869 alr2269

21 90

127 356

alr1819

58

147

alr2489 alr1819

21 58

68 235

alr2269

90

214

alr2489 alr1819

21 58

100 594

alr2269

90

493

alr2489 alr1819

21 58

105 990

alr2269

90

294

8

omp85

250 omp85

9

230 omp85

10

180 omp85

11

160

alr1819

58

970

12

140

alr1819

58

1250

13

100

alr1819

58

1030

nrtA

alr0608

49

920

schT

alr0397

94

77

(Putative) function porin porin porin porin porin TolC porin porin porin TolC porin b-Barrel protein assembly ATP-dependent protease porin ATP-dependent protease b-Barrel protein assembly Cell surface protein Cell surface protein b-Barrel protein assembly Cell surface protein b-Barrel protein assembly Cell surface protein b-Barrel protein assembly Cell surface protein Cell surface protein Cell surface protein Nitrate transport protein Iron transport

The identified proteins correspond to the marked bands (white arrowheads) found by histidine- and deoxycholate-based native (HDN-) PAGE analyses (Figure 3b) from top to bottom. a Estimated molecular weight in kDa. b Molecular weight predicted. c Mascot score. d Contaminated with the identified photosystem I and photosystem II complex.

200 kDa we did not detect Toc34, which is consistent with the immunodecoration. Interestingly, the 220 kDa complex contained Toc159, Toc75-III, Toc75-V and Toc64 (Table 4). Toc75-V, also termed Oep80, is thought to possess a function comparable to that of Sam50 and thus different from pre-protein import (Schleiff and Becker, 2011), and Toc64 is a low and dynamically associated component of the TOC complex, which was proposed to interact via Toc34 with the TOC complex (Sohrt and Soll, 2000; Qbadou et al., 2007). The 200 kDa band contained the two Toc75 proteins, Toc75-III and Toc75-V, as well as the ATP-dependent outer envelope channel Oep21 (e.g. Bo¨lter et al., 1999). Whether the composition of the two latter bands indeed reflects one complex each or co-migrating complexes has to be further investigated. Summarizing, we observed three distinct states of the TOC core complex, namely the once containing all three components, one containing Toc75-III and Toc159 and one with Toc75-III only. Thus, in future it should be possible to investigate the assembly of this complex by the native PAGE introduced in here. Analysis of complexes in chloroplast inner envelope membranes by native PAGE In the inner envelope we detected seven Coomassie stainable complexes by HDN-PAGE analysis when solubilizing the membranes with digitonin (Figure 4b). These complexes migrated with an approximate molecular mass of 100, 110, 130, 200, 240, 530 and 700 kDa (Figure 4b, left, lane 5, 6). Using n-decyl-b-maltoside (DM, lane 1, 2) or DDM (lane 3, 4) we observed slight differences in the migration of small molecular mass complexes and an additional complex of >800 kDa. Using Tic110 antibodies we could assign some of the Coomassie stainable complexes to the TIC translocon, which were subsequently analyzed in more detail (Figure 5). Using specific antibodies against Tic110, Tic55 and Tic22 we observed distinct complexes by HDN-PAGE. One complex migrated at 250 kDa and contained Tic110 and Tic55, as previously described (Ku¨chler et al., 2002). Three other complexes migrating at 400, 230 and 100 kDa were stained by Tic110 antibodies, whereas the complex migrating at 550 kDa was immunodecorated by Tic22 antibodies only (Figure 5). To confirm these observations we analyzed the protein composition of these complexes by mass spectrometry (Table 5). We could not detect Tic22, but in the complex migrating at 550 kDa we detected Tic20, which has previously been shown to assemble this complex (Kouranov and Schnell, 1997). Consistent with the results observed by western blot we detected Tic110 in the 350, 250 and 230 kDa bands. Remarkably, the 250 and 230 kDa complexes contained ClpC in addition to Tic55, confirming interactions between these proteins reported earlier (e.g. Akita et al., 1997; Nielsen et al., 1997). However, this questions the


HDN-PAGE for membrane protein analysis 189

Figure 4. Native PAGE analysis of complexes in the chloroplast outer and inner envelope membrane of pea (Pisum sativum). Equal amounts of outer (a) and inner (b) chloroplast envelope membrane vesicles were solubilized with n-decyl-b-maltoside (lanes 1 and 2; DM), dodecyl maltoside (lanes 3 and 4; DDM) and digitonin (lanes 5–7; Dig) at concentrations as indicated and the complexes were separated by the three native gel systems (histidine- and deoxycholate-based native, HDN, left; blue native, BN, middle; high-resolution clear native, hrCN, right). The samples of the 2% digitonin-solubilized sample were blotted and immunodecorated with aToc34 and aTic110, respectively. The molecular masses of the standard used are indicated on the left of each panel. Arrowheads point to analyzed bands (Tables 4 and 5). The Rubisco complex (confirmed by mass spectrometry, not shown) is indicated by a star.

stoichiometry of this complex, which at best could be a 1:1:1 complex. The complexes with lower molecular masses contained several transporters such as the glucose, SAM or PEP transporter. In the high molecular mass complexes we observed Ycf1, Ycf2 and a malate dehydrogenase, but not Tic20 or Tic21 as previously reported for high molecular mass complexes (Kikuchi et al., 2009). The latter was detected at 135 kDa only. However, we cannot exclude that we missed these proteins due to their low abundance. DISCUSSION We have presented HDN-PAGE as a new version of native PAGE which shows properties comparable to conventional BN-PAGE for the analysis of mitochondrial or thylakoid systems, but exhibits superior performance when analyzing complex membrane fractions such as cyanobacterial cell walls or the chloroplast envelope membranes. The power of HDN-PAGE as a new analytical tool was demonstrated by

isolation of individual protein complexes from thylakoid membranes of Anabaena sp. PCC 7120 and inner envelope membranes of P. sativum and subsequent analysis by mass spectrometry. It is not entirely clear why HDN-PAGE showed better performance than BN-PAGE when isolated membrane fractions were analyzed. Niepmann and Zheng (2006) who invented the 2-amino-2-(hydroxymethyl)-1,3-propanediol (TRIS)-histidine buffer system speculated that it might be especially suitable for the separation of basic proteins. Since the pH of the HDN-PAGE buffers is the same as in the TRIShistidine system this holds true. Additionally the obligate use of deoxycholate also confers to basic proteins a negative net charge, improving their resolution. It appears that the application of a discontinuous gel system employs a shift from pH 8 to >8 that speeds up the trailing ion histidine which has a tremendous impact on resolution. The ion gradient formed in the stacking gel then causes the protein


190 Roman Ladig et al. Table 4 Mass spectrometric analysis of pea chloroplast outer envelope membrane complexes

Table 5 Mass spectrometric analysis of pea chloroplast inner envelope membrane complexes

Spot

MWa

Source

Name

Gene

MSC

Spot MW Source Name

Gene

1

700

Dig

AtCG01130 AtCG01280

186 143

Dig

2

700

Dig

At3G47520 AtCG01280

111 282

3

250

Dig

3

550

DM

At5G24650

196

4

220

Dig

4 5

350 250

DM Dig

6

230

DM

5

200

Dig

431 175 112 354 281 111 253 493 93 157 717 203 93 851 122 35

>800 Dig

500

At4G02510 At3G46740 At1G02280 At4G02510 At3G46740 At1G02280 At4G02510 At3G46740 At1G02280 At4G02510 At3G46740 At3G17970 At5g19620 At3G46740 At5G19620 At1G20810

1

2

Toc159 Toc75-III Toc34 Toc159 Toc75-III Toc34 Toc159 Toc75-III Toc34 Toc159 Toc75-III Toc64-III Toc75-V Toc75-III Toc75-V Oep21

7

200

Dig

8

135a DM

The identified proteins correspond to the marked bands (white arrow heads) found by histidine- and deoxycholate-based native (HDN-) PAGE analyses of Figure 4a, from top to bottom. a The estimated molecular weight of the complex migration in kDa (MW). Listed are the detergent used for solubilization (source), the name of the identified protein (Name), the gene number of the respective A. thaliana gene used for annotation (Gene), and the mascot score (MSC). Dig, digitonin.

9

10

11

Figure 5. Western blot of translocon in the inner envelope membrane of chloroplasts (TIC) complexes in the chloroplast inner envelope membrane of pea (Pisum sativum). Inner envelope membranes were solubilized by 2% (w/v) n-decyl-b-maltoside (DM, lanes 1, 3 and 5) or Dig (digitonin, lanes 2, 4 and 6) and separated as in Figure 4 followed by western blotting using antibodies as indicated.

complexes to focus in a sharp band. A major difference from other samples is the protein to lipid ratio that is rather high in mitochondria and thylakoid membranes but rather low in the cell wall or the envelope membranes. Furthermore, the presence of the DDM and DOC mixture in the cathode buffer

130

110

80

Dig

Dig

Dig

Ycf1 AAA-type ATPase family protein Ycf2 Malate dehydrogenase AAA-type ATPase family protein Ycf2 Tim17/Tim22/Tim23 family protein Tic110 Tic110 CLPC Tic55-II Tic110 CLPC Tic55-II Hydroperoxide lyase 1 Tim17/Tim22/Tim23 family protein Tic32 Inner envelope protein 37 Unknown Unknown Hydroperoxide lyase 1 Tic32 Inner envelope protein 37 Major facilitator protein Chloroplast ATP/ADP translocator Inner envelope protein 37 PEP translocator Unknown Glucose transporter 1 SAM transporter Putative membrane protein Inner envelope protein 37 Unknown Unknown Cell growth defect factor 1

MSC

At1G06950 1420 At1G06950 544 At5G50920 175 At2G24820 157 At1G06950 549 At5G50920 146 At2G24820 101 At4G15440 201 At5G24650 164 At4G23420 At3G63410

101 320

At5G08540 At5G12470 At4G15440 At4G23420 At3G63410

338 219 137 107 473

At5G59250

208

At1G80300

193

At3G63410

514

At5G33320 At5G08540 At5G16150 At4G39460 At1G32080 At3G63410

135 306 167 140 125 547

At5G12470 At5G08540 At5G23040

354 153 120

The identified proteins correspond to the marked bands (white arrow heads) found by histidine- and deoxycholate-based native (HDN-) PAGE analyses (from top to bottom). Listed is the number of the complex analyzed according to Figure 4b (labeled from top to bottom), the estimated molecular weight of the complex migration in kDa (MW), the detergent used for solubilization (source), the name of the identified protein (Name), the gene number of the respective Arabidopsis thaliana gene used for annotation (Gene), and the mascot score (MSC). Please note, only candidates with MSC higher than 100 and detected in two independent samples are shown. a In this lane Tic21 was detected with an MSC of 65. Abbreviations: Dig, digitonin; DM, n-decyl-b-maltosi.


HDN-PAGE for membrane protein analysis 191 (Table 1) seemed to be advantageous, as also hrCN-PAGE to some extent performed better for all three isolated membrane systems than the BN-PAGE system (Figures 3 and 4). This effect may be explained by dilution of lipids with short chain fatty acids resulting in a better resolution. Additionally, as discussed, the decreased lipid to protein ratio of the cell wall and chloroplast envelope fractions might explain the differential effects observed. Finally, the discontinuous native PAGE system in combination with a mild charge-conferring detergent mix may allow for immediate and fast migration of low molecular weight components like lipids, further improving resolution performance. On the other hand, the improved capacity of HDN-PAGE to deal with a high lipid content seems to result in a somewhat stronger dissociating effect with membranes with a larger fraction of proteins. This was evident from the absence of supercomplexes and partial dissociation of wild-type complex I after HDN-PAGE of mitochondrial membranes from Y. lipolytica. Overall, our study demonstrates that the choice of the right native-PAGE system is critical for optimal separation of membrane-bound multiprotein complexes. The HDNPAGE system presented here seems to be particularly suited to the analysis of protein complexes in isolated membranes exhibiting a high lipid to protein ratio. As such it complements previously available protocols and broadens the usefulness of native-PAGE approaches to analyse macromolecular protein complexes in biological membranes. EXPERIMENTAL PROCEDURES Materials Coomassie Brilliant Blue G-250, bovine serum albumin and digitonin were purchased from Serva (http://www.serva.de/ enDE/index.html), DDM and DM from Biomol (http://www. enzolifesciences.com/) or Glycon (http://www.glycon.de/), respectively. AEBSF and E-64 were purchased from Applichem (http:// www.applichem.com/). Micrococcal nuclease was purchased from Fermentas (http://www.fermentas.com/). Percoll was purchased from Sigma (http://www.sigmaaldrich.com/). Miracloth was purchased from Calbiochem (http://www.merck-chemicals.de/). All other chemicals were bought from Carl Roth GmbH (http:// www.carlroth.com/). Native molecular weight standards (HMW Native Marker kit) are purchased from GE Healthcare (http:// www.gehealthcare.com/).

Isolation of mitochondria from bovine hearts and Y. lipolytica Bovine heart mitochondria were prepared according to Smith (1967). Yarrowia lipolytica were grown as described (Kashani-Poor et al., 2001), cells were broken using a glass bead mill cell disintegrator (Euler Biotechnology, http://www.biotechnologie-euler.de/) and mitochondria were isolated as described in Kashani-Poor et al. (2001).

Isolation of pea chloroplasts Pisum sativum (var. golf) was grown for 7–8 days and Medicago sativa for 14 days under greenhouse conditions. Fifty grams of leaves was harvested and homogenized in 400 ml of 50 mM HEPES-KOH, pH 7.8, 330 mM sorbitol, 2 mM EDTA, 1 mM MnCl2 and 1 mM MgCl2. After filtration through Miracloth (Calbiochem) 5 mM of fresh prepared DTT (final) was added and the mixture was centrifuged for 2 min at 1500 g. The pelleted chloroplast fraction was resuspended in and layered on top of a two-step Percoll (Sigma) gradient (80% v/v and 40% v/v), centrifuged for 10 min at 10 000 g in a swing bucket rotor and the chloroplasts were collected from the 40/80% interface. After washing of chloroplasts twice they were stored on ice in the dark and chlorophyll content was measured according to Arnon (1949). Isolation of outer and inner envelope membranes was performed according to the established protocols (Schleiff et al., 2003).

Preparation of Anabaena thylakoids and cell wall fractions Preparation of the Anabaena sp. PPC 7120 cell wall and thylakoids was performed according to Nicolaisen et al. (2009a,b). In brief: cells were grown in 1 L of BG11 medium and bubbled with air containing 1% v/v CO2 to a concentration of about OD750 = 2. Cells were collected at 4000 g for 5 min at room temperature (22C). Cell sediments were washed once in Buffer A [5 mM HEPES pH 8.0 and 1 mM phenylmethylsulfonyl fluoride (PMSF)]. After washing, cells were re-collected, resuspended in 30 ml of Buffer A, supplemented with protease cocktail inhibitor tablets (Roche, http://www.roche. com/) and 1 mM of PMSF to prevent protein degradation. Cell suspensions were lysed by French pressing twice at 1000 p.s.i. All the following steps were performed on ice. Broken cells were centrifuged at 48 000 g for 45 min at 4C; the upper dark green layer was carefully resuspended in 55% w/v sucrose in Buffer B (20 mM HEPES pH 8.0 and 0.2 mM PMSF). Six floating sucrose density gradients were prepared by adjusting the sucrose concentration of the cell suspension to 55% and overlaying the mixture with 40, 30 and 10% sucrose solutions of Buffer B. The cell fractions were separated by centrifugation at 130 000 g for 16 h at 4C. Green thylakoid membrane layers at the 55–40% sucrose interface were collected and diluted with washing buffer containing 20 mM HEPES pH 8.0 and 1 mM PMSF. Thylakoid membranes were collected at 130 000 g for 1 h at 4C. Membranes were resuspended in minimal volume of Buffer B. Thirty micrograms of thylakoid membranes (determined by Bradford protein assay) was 30 min solubilized in 30 ll of sample buffer containing 1.5% digitonin and centrifuged at 50 000 g for 30 min at 4C; supernatant was subjected to native PAGE analysis.

Native PAGE analysis of membrane fractions – electrophoresis The use of native PAGE has been amply described (e.g. Scha¨gger and von Jagow, 1991; Scha¨gger et al., 1994; Niepmann and Zheng, 2006; Wittig et al., 2006; Reisinger and Eichacker, 2007; Wittig and Scha¨gger, 2008). The composition of the different buffer systems is given in Table 1. Please note that when using highly concentrated TRIS buffer stocks it is critical to control pH before use since the pH is temperature dependent. Here, 20% glycerol was used for gradient formation. All gels were prepared in a cold room with pre-cooled solutions. The HDN-PAGE gels were cast without stacking gel with a 3.5–12% (v/v) acrylamide gradient from bottom to top with a Hoefer gradient mixer (GE Healthcare). Gels for BN-PAGE and hrCN-PAGE were prepared in the same way with a 4–12% (v/v) acrylamide gradient separation gel and a 4% (v/v) acrylamide stacking gel.


192 Roman Ladig et al. Gels and electrophoresis units were pre-cooled and run in the cold (4C) overnight. The initial voltage was set to 50 V and raised to a maximum of 200 V to finish the run (for 0.15 · 16 · 18 cm gels).

Native PAGE analysis of membrane fractions – sample preparation Solubilization of organelles and membrane vesicles was performed with buffers as stated in Table 1. All steps were performed on ice (