- Email: [email protected]
Mitochondrial F1F0-ATP synthase and organellar internal architecture
The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Review
Mitochondrial F1 F0 -ATP synthase and organellar internal architecture Jean Velours ∗ , Alain Dautant, Bénédicte Salin, Isabelle Sagot, Daniel Brèthes Université Bordeaux 2, CNRS, Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint Saëns, 33077 Bordeaux Cedex, France
a r t i c l e
i n f o
Article history: Available online 24 January 2009 Keywords: Yeast Mitochondria ATP synthase Supramolecular species
a b s t r a c t The mitochondrial F1 F0 -ATP synthase adopts supramolecular structures. The interaction domains between monomers involve components belonging to the F0 domains. In Saccharomyces cerevisiae, alteration of these components destabilizes the oligomeric structures, leading concomitantly to the appearance of monomeric species of ATP synthase and anomalous mitochondrial morphologies in the form of onion-like structures. The mitochondrial ultrastructure at the cristae level is thus modified. Electron microscopy on cross-sections of wild type mitochondria display many short cristae with narrowed intra-cristae space, whereas yeast mutants defected in supramolecular ATP synthases assembly present a low number of large lamellar cristae of constant thickness and traversing the whole organelle. The growth of these internal structures leads finally to mitochondria with sphere-like structures with a mean diameter of 1 m that are easily identified by epifluorescence microscopy. As a result, ATP synthase is an actor of the mitochondrial ultrastructure in yeast. This paper reviews the ATP synthase components whose modifications lead to anomalous mitochondrial morphology and also provides a schema showing the formation of the so-called onion-like structures. © 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Yeast strains and plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biochemical procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fluorescence microscopy analyses of yeast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Ultrastructural studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ATP synthase is an actor of mitochondrial ultrastructure in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The formation of mitochondrial onion-like structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The yeast mitochondrial F1 F0 -ATP synthase is a large 600 kDa complex that contains at least 17 distinct subunits (Velours and Arselin, 2000) organized into a catalytic part called F1 and a base piece called F0 . F0 is embedded in the mitochondrial membrane and is mainly composed of hydrophobic subunits forming a specific proton channel. The enzyme uses the proton electrochemical
Abbreviations: BN-PAGE, blue native polyacrylamide slab gel electrophoresis; F1 and F0 , peripheral and integral membrane portions of ATP synthase. ∗ Corresponding author. Tel.: +33 556999001; fax: +33 556999010. E-mail address: [email protected] (J. Velours). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.01.011
1783 1784 1784 1784 1784 1784 1784 1784 1786 1788 1789
gradient generated by the respiratory chain to produce ATP from ADP and inorganic phosphate. This enzyme is a molecular rotary motor where the proton translocation through F0 induces the rotation of a ring of 10 hydrophobic subunits (subunit 9 in yeast). This drives the rotation of a central stalk inside the catalytic head that emerges from the membrane (Devenish et al., 2008 for review). The mitochondrial ATP synthase adopts supramolecular structures that have been found in a large range of organisms by native gel electrophoresis, mainly in dimeric forms (Arnold et al., 1998; Eubel et al., 2003; Krause et al., 2005) but also in higher molecular forms (tetramers, hexamers) according to the electrophoresis technique used (Paumard et al., 2002; Giraud et al., 2002; Krause et al., 2005; Wittig et al., 2008). Depending on the detergent utilized during the extraction and the purification procedures, the
1784
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
isolated complexes were monomers and dimers as shown by electron microscopy single particle analysis (Rubinstein et al., 2003; Lau et al., 2008; Dudkina et al., 2005, 2006; Minauro-Sanmiguel et al., 2005; Thomas et al., 2008), although higher structures such as tetramers have also been reported (Thomas et al., 2008, supplementary data). The isolated ATP synthase dimers adopt a V-like structure with the two F0 parts linked together. Several F0 subunits are essential for dimerization. In Saccharomyces cerevisiae, it has been shown that subunits e and g that are not involved in the ATPase or ATP synthase activity are essential actors for dimerization (Arnold et al., 1998) and oligomerization (Fronzes et al., 2006). These two proteins are present only in mitochondria. Other components such as subunit 4, the homologous subunit to the bsubunit of beef ATP synthase, and other proteins of the peripheral stalk were found to be involved in vivo in the interactions between monomers (Gavin et al., 2005; Fronzes et al., 2006; Wittig and Schägger, 2008). Supramolecular structures of mitochondrial ATP synthase (tetramers, hexamers) have been reported both in blue native gel experiments and in cross-link experiments showing that subunits e and g are involved in dimer stabilization (Fronzes et al., 2006) and in the oligomerization process (Arselin et al., 2003; Krause et al., 2005; Wittig and Schägger, 2005; Bustos and Velours, 2005). Such assemblies have been observed in situ by atomic force microscopy (Buzhynskyy et al., 2007), by transmission electron microscopy (Thomas et al., 2008) and by electron cryo-tomography (Strauss et al., 2008). Originally discovered in Paramecium multimicronucleatum mitochondria (Allen et al., 1989), the supramolecular organization of ATP synthases associates the linear and regular arrays of dimer assembly on cristae to the formation of tubular cristae (Allen, 1995). Surprisingly, yeast mutant mitochondria devoid of either subunit e or g display numerous digitations and onion-like structures, thus suggesting a relationship between ATP synthase dimerization/oligomerization and cristae morphology (Paumard et al., 2002; Soubannier et al., 2002), in agreement with the hypothesis of Allen (1995). This observation was reinforced by the aberrant mitochondrial morphology in cells whose ATP synthase complexes were cross-linked in vivo through subunit ␥-27-DsRed fusion proteins (Gavin et al., 2004). Thus it appears that by promoting supramolecular structures, ATP synthase is one of the numerous actors involved in the establishment of the internal mitochondrial morphology (Mannella, 2006; Zick et al., 2009 for reviews). Here we summarize the alterations in ATP synthase subunits of yeast that lead to mitochondria showing onion-like structures and provide data describing the formation of such objects. 2. Methods 2.1. Yeast strains and plasmids The Saccharomyces cerevisiae strain D273-10B/A/H/U (Mat ␣, met6, ura3, his3) was the wild type strain (Paul et al., 1989). The TIM11 and ATP20 strains were constructed by a PCR-based mutagenesis (Güldener et al., 1996). The strains containing modified versions of subunits e and g were obtained by integration of the mutated versions of TIM11 and ATP20 genes at the chromosomic locus in the respective deleted-disrupted strains (Arselin et al., 2004; Bustos and Velours, 2005). The tetO-TIM11 and tetO-ATP20 strains were constructed as described in Arselin et al. (2004). 2.2. Biochemical procedures Cells were grown aerobically at 28 ◦ C in a complete liquid medium containing 2% lactate as carbon source (respiratory medium) or 2% galactose as indicated. The rho- cell production in
cultures was measured on glycerol plates supplemented with 0.1% glucose in the presence or not of 20 g/ml of doxycycline. Electrophoretic and western blot analyses of mitochondrial proteins are described in Arselin et al. (2004). 2.3. Fluorescence microscopy analyses of yeast cells For visualization of mitochondria by fluorescence microscopy, a pRS303 vector bearing the wild type ATP20 gene under the dependence of a tetracycline-regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase was integrated in the his3 gene of cells devoid of subunit g. The resulting strain was grown for 6 h with 2% galactose as carbon source with or without 20 g/ml of doxycycline. The cells were fixed by formaldehyde addition (3.7% final) directly in the culture. The cells were observed in a fully automated Zeiss 200 M inverted microscope (Carl Zeiss, Thornwood, NY) equipped with an MS-2000 stage (Applied Scientific Instrumentation, Eugene, OR), a Lambda DG4 LS 175-watt xenon light source (Sutter, Novato, CA), a 100 × 1.4 numerical aperture plan-apochromat objective. The filter cube used was an Endow GFP long pass (excitation: HQ470/40, emission: HQ500lp, and beam splitter: Q495lp–Chroma Technology Corp. Rockingham, VT). Images were acquired using a CoolSnap HQ camera (Roper Scientific, Tucson, AZ). The microscope, camera, and shutters (Uniblitz, Rochester, NY) were controlled by SlideBook software (Intelligent Imaging Innovations Inc., Denver, CO). All the pictures are maximum projection of z-stacks acquired with a Z step of 0.2 m. All the image measurements were done using the SlideBook 4.1. software (Intelligent Imaging Innovations Inc., Denver, CO). 2.4. Ultrastructural studies Freezing and freeze substitutions of yeast cells pellets were performed as described in Paumard et al. (2002). 3. Results and discussion 3.1. ATP synthase is an actor of mitochondrial ultrastructure in yeast Our laboratory has shown by electron microscopy that the loss of supramolecular structures of ATP synthases leads to anomalous mitochondria. This anomaly was found in the null mutant in TIM11 and ATP20 genes encoding for subunits e and g, respectively (Paumard et al., 2002), two components associated with ATP synthase and that are involved in the dimerization of the enzyme (Arnold et al., 1998) but not in its activity. The main observation obtained by transmission electron microscopy was the presence of mitochondria sometimes having sphere-like structures with a diameter of 0.8–1 m. These objects are composed of several dense layers separated by white spaces (Paumard et al., 2002). By immune-electron microscopy, the porin protein, which is a standard outer membrane indicator, was found to be restricted to the outer-most membrane, whereas ATP synthase was associated with the inner membranes. Thus, the dense layers correspond to the matrix space whereas the white spaces correspond to the inner membrane space. The abnormal mitochondrial structure was interpreted as the consequence of an uncontrolled biogenesis of the inner mitochondrial membrane. The alterations described above were observed in mutant cells grown for at least 30 generations after deletion of the genes encoding subunits e or g. However, 40% of TIM11 and ATP20 mutant cells spontaneously converted to rho- cells during cultures (Fig. 1), whose effect was to increase the doubling time with nonfermentable medium and to decrease oligomycin sensitivity of ATP synthase. Rho- cells that are altered in their mitochondrial DNA
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
1785
Fig. 1. Appearance of rho- cells upon depletion of subunits e or g. The amount of subunits e and g was modulated as a function of time by using a doxycycline-regulated expression system. Aliquots of cultures were plated to measure the amount of rhocells that were spontaneously generated. e and g refer to strains whose nuclear genes TIM11 and ATP20 encoding the subunits e and g, respectively were inactivated. (1) (tetO-TIM11 cells), (2) (tetO-ATP20 cells). wt, wild type.
have no F0 domain and therefore contain free F1 in the matrix with ATPase activity insensitive to oligomycin. Such a conversion in rhocells was always observed upon modification of genes encoding subunits of yeast ATP synthase (Contamine and Picard, 2000). As
Fig. 3. The lack of ATP synthase subunits e and g leads to an anomalous mitochondrial structure. (A and B) Transmission electron microscopy of tetO-TIM11 cells grown in a respiratory medium without (A) or with doxycycline (B) for 12 h. m = mitochondria. The bar indicates 500 nm.
Fig. 2. The decrease in the amount of subunit e leads to the lack of subunit g and a concomitant decrease in the amount of oligomeric species of yeast ATP synthase. (A and B) Western blot analyses of mitochondria isolated from tetO-TIM11 cells grown in a respiratory medium in the presence of 20 g/ml of doxycycline for 2 and 6 h. The blots were probed with polyclonal antibodies raised against either subunits e and i (A) or subunits g and i (B). (C) BN-PAGE analysis of digitonin extracts of mitochondria isolated from yeast cells grown in the presence of doxycycline for 2, 6 and 12 h. The slab gel was stained with Coomassie brilliant blue. wt = wild type, dox. = doxycycline, oli., dim., and mon. = oligomeric, dimeric and monomeric species of yeast ATP synthase, respectively. The star indicates the location of the (III + IV)2 supercomplex.
a result, it was important to differentiate the effect of the lack of subunits e or g on mitochondrial morphology from that due to spontaneous conversion in rho- cells. To this end, the expression of the TIM11 and ATP20 genes was modulated by using a tetracyclineregulatable promoter system (Garí et al., 1997; Arselin et al., 2004). During the time course of the depletion in either subunit e or g in tetO-TIM11 or tetO-ATP20 cells, the amount of rho- cells remained comparable to that of wild type cells. A small increase in the rho- cell content was noted only after 20 h of culture in the presence of doxycycline (Fig. 1). As a result, the mutant strains were grown for up to 12 h in the presence of the antibiotic during the following experiments. Doxycycline addition to the culture medium of tetO-TIM11 cells led to a rapid decrease in amounts of subunit e and subunit g (Fig. 2A and B). Indeed, it was previously reported that the lack of subunit e induces a loss of subunit g (Arnold et al., 1998). The loss of the two subunits led to the disappearance of the supramolecular forms of the ATP synthase in digitonin extracts (Fig. 2C), which correlates with the change in mitochondrial morphology (Fig. 3), as shown by the presence of onion-like structures already described in the null mutant in genes encoding subunits e and g. The behavior of the mitochondrial network in such mutants was also examined. To this end, a pRS303 vector bearing the wild type ATP20 gene under the dependence of a tetracycline-
1786
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
Table 1 Yeast mutants altered both in ATP synthase ability to promote supramolecular structures and in mitochondrial morphology. Strains
Modification
References
TIM11 ATP20
Disruption of TIM11 gene Disruption of ATP20 gene
Paumard et al. (2002) Paumard et al. (2002)
eG19L e19A eG15L
Alteration of GxxxG motif of subunit e
Arselin et al. (2003)
eK32 stop gV100 stop gR106 stop
Deletion of C-terminal part of subunit e Deletion of C-terminal part of subunit g Deletion of C-terminal part of subunit g
Unpublished data Bustos and Velours (2005) Bustos and Velours (2005)
gG101L gG105L g102A
Alteration of GxxxG motif of subunit g
Bustos and Velours (2005)
4TM1 4(49–55) RFY5-1 tetO-TIM11 tetO-ATP20 ␥-27-DsRed
Deletion of first 43 amino acid residues of subunit 4 Deletion in loop 49–55 of subunit 4 Controlled depletion of subunit h Controlled depletion of subunit e Controlled depletion of subunit g DsRed attached to the C-terminus of subunit ␥
Soubannier et al. (2002) Weimann et al. (2008) Goyon et al. (2008) Arselin et al. (2004) Arselin et al. (2004) Gavin et al. (2004)
regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase was integrated in the his3 gene of cells devoid of subunit g. Cells were grown for 6 h with 2% galactose as carbon source with or without doxycycline and observed by epifluorescence microscopy. In the absence of doxycycline, a typical elongated and branched mitochondrial network was present (Fig. 4). After 6 h of growth in the presence of the antibiotic, the tubular mitochondrial network changed to punctuated roundedshape organelles. Indeed a careful analysis along the z-axis showed that these objects are hollow spheres probably corresponding to the mitochondria with onion-like structures found by transmission electron microscopy. The mean diameter of these objects is 1.05 ± 0.4 m (175 objects measured) and their mean number per cell is 2.43 ± 1.89 (72 cells were scored). The size of these objects is in agreement with that observed by transmission electron microscopy. These data clearly indicate that the yeast mitochondrial network is strongly altered in the absence of subunit g. As a consequence, the lack of supramolecular structures of ATP synthase might be considered as one of the parameters influencing mitochondrial morphology. Since the first experiments where subunit e or g was removed from the enzyme, new data have reinforced the link between the ability of ATP synthase to dimerize and the altered morphology.
Table 1 summarizes alterations in ATP synthase subunits that lead both to the absence of ATP synthase dimerization and altered mitochondrial morphology. Subunits e and g display a conserved GxxxG dimerization motif in the hydrophobic part of the sequence. The glycine residues were replaced by leucine to increase the steric hindrance and alternatively an alanine residue was inserted after the first glycine in the GxxxG motif. All these alterations lead to the lack of ATP synthase dimerization and to the presence of mitochondria with onion-like structures (Arselin et al., 2003; Bustos and Velours, 2005). Subunit 4, the homologous protein to the b-subunit, is also involved in the dimerization process. The eukaryotic mitochondrial protein displays a transmembranous domain in its N-terminus composed of two hydrophobic segments. The removal of the first 43 amino acid residues containing the first membrane-spanning segment did not alter the function of ATP synthesis, but led to the lack of subunit g and as a consequence to the loss of supramolecular structures for the ATP synthase and the appearance of onion-like structures (Soubannier et al., 2002). From these data and crosslinking experiments, we hypothesized that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the gsubunit that is an associated ATP synthase component only present in the mitochondrial enzyme. In addition, the shortening of loop 47–55 linking the two membrane-spanning segments of subunit 4 significantly alters the oligomerization of the yeast ATP synthase and as a consequence the mitochondrial morphology (Weimann et al., 2008). Another question is the ultrastructure of isolated mitochondria from cells showing anomalous mitochondrial morphologies. Like isolated wild mitochondria, mitochondria devoid of either subunit e or g are also circular but display numerous membranes in the matrix space that traverses the organelle (Arselin et al., 2004). Indeed, the aspect of isolated mitochondria is certainly far from reality and probably results from alterations in the ultrastructure of organelles during isolation. 3.2. The formation of mitochondrial onion-like structures
Fig. 4. The absence of ATP synthase subunit g alters the mitochondrial network. The construction of the strain harboring the wild type ATP20 gene under the dependence of a tetracycline-regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase is described in Section 2. Cells were grown for 6 h in a galactose medium as carbon source in the presence or not of 20 g/ml of doxycycline and examined by epifluorescence microscopy. The bar indicates 2 m.
An open question is the formation of onion-like structures that display successive layers of membranes. Fig. 5 shows wild type yeast mitochondria in the cellular context. With our sample preparation procedure (Paumard et al., 2002), the cristae of the wild type cells are small with membranes joined in such a way that the intra-cristae space is not visible. In Fig. 6A and B, the mitochondrial morphology of the mutant eG15L is shown as an example. The mutation alters the conserved GxxxG domain
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
1787
Fig. 5. Wild type mitochondria have short cristae and a narrow intracristae space. Wild type yeast cells were grown in a respiratory medium and prepared for transmission electron microscopy as in (Paumard et al., 2002). The arrows indicate cristae. The bar indicates 100 nm.
described above and as a consequence prevents ATP synthase dimerization/oligomerization. Transmission electron microscopy of the eG15L mutant exemplifies a typical organellar ultrastructure. The mitochondria appear elongated. Fig. 6A shows mitochondria with unique lamellar cristae whose thickness is evaluated at 30 nm. The two cristae membranes delimit a 12 nm white space that is nearly constant over almost all the organelle except at the cristae junction level, where a thin 3 nm channel is observed. The compartments were identified by immunogold labeling (Paumard et al., 2002). The two dense layers on both sides of the cristae correspond to the matrix space and the white space corresponds to the intracristae compartment. The outer membrane lies at the periphery of the organelle and is probably joined with the inner peripheral membrane. Other mitochondria display two (Fig. 6B) and sometimes three (not shown here) lamellar cristae. In all the samples, the main point is the presence of lamellar structures traversing the organelle when the ATP synthase lacks its capacity to oligomerize. Indeed, intermediary organelles between “normal” and onion-like mitochondria can exist. An example is given with mutant 4(49–55). In this strain the loop linking the two membrane-spanning segments of subunit 4 was shortened. Despite the presence of subunits e and g, most of ATP synthases showed as monomers on CN-PAGE analysis of digitonin extracts (Weimann et al., 2008). The cristae are frequently pinched in this mutant (arrows in Fig. 6C) and the intermembrane space appears larger than that of wild-type cristae. The formation of onion-like structures was interpreted as an uncontrolled biogenesis of inner membrane sheets, which promotes mitochondrial elongation and leads to large organelles inside the cells. Transmission electron micrographs (Fig. 7) show the formation of onion-like structures in a cell progressively devoid of either subunit e or g. The decrease in the expression of subunit e leads to spherical mitochondria (left of the figure) with several cristae that traverse the mitochondria (corresponding to 4 h of growth with doxycycline). Note the large intra-cristae space that differs significantly from that of wild type mitochondria. The growth of several lamellar sheets of the inner membrane enlarges
Fig. 6. Anomalous structure of mitochondrial cristae in yeast mutants devoid of supramolecular structures of ATP synthase. eG15L mutant (A and B) and 4(49–55) (C). Cells were grown in a respiratory medium and prepared for transmission electron microscopy as in Paumard et al. (2002). The insert enlarges a section of the inner structure of mutant mitochondria. The arrows indicate spots where membranes of cristae are joined. The bar indicates 500 nm.
the organelle and gives the aspect of a saucer and open or closed spherical structures as seen by electron microscopy, depending on the cutting plane of the samples (corresponding to 12 h of growth with doxycycline). The size of the objects is in agreement with that observed by epifluorescence microscopy. The anomalous structures are reversible. Using the doxycycline-regulatable expression system, Arselin et al. (2004) showed that onion-like structures are formed after five generations. When doxycycline is removed from the culture medium, normal cristae are observed after five generations, corresponding to a recovery of both subunits e and g and supramolecular forms of ATP synthase. Several points emerge from these data. In mutants devoid of supramolecular associations of ATP synthase, the ultrastructure of cristae is drastically modified. Whereas wild type cristae are short, mutant cristae traverse the whole organelle longitudinally and the intra-cristae space is drastically increased (12 nm). This indicates that the intra-cristae space is able to move from the closed state observed in wild type mitochondria (Fig. 5) to the relaxed state of onion-like structures (Fig. 6). The constant size of the intra-cristae space in mutant cells suggests the existence of structures inside this space that keep the distance constant between the two cristae membranes. However, these structures are probably lost during
1788
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
Fig. 7. Representation model of the formation of yeast mitochondrial onion-like structures during depletion of subunit e or g (→) and their visualization by electron microscopy according to the cutting plane of the samples (- - ->). wt = wild type. The 3D model of objects was created by using Google Sketchup.
the isolation of mitochondria since the cristae appear larger and swollen except at the cristae junction (Arselin et al., 2004). Electron tomography of isolated yeast wild type mitochondria demonstrated a three-dimensional network of large lamellae that were interconnected in some places (Thomas et al., 2008). In addition, transmission electron microscopy of negatively stained submitochondrial particles revealed the presence of two parallel rows of ATP synthase along the membrane border with a density of 7–10 pairs of particles for 100 nm (Thomas et al., 2008). Similar data were obtained by cryo-electron tomography of bovine heart mitochondria where rows of dimers were found in the most tightly curved membrane regions of cristae (Strauss et al., 2008). According to Allen (1995), ATP synthase oligomerization could be responsible for the tubulation of the mitochondrial inner membrane allowing the formation of cristae, which in fine increases the surface of the inner membrane available for components of oxidative phosphorylation. Since the tomographic analysis of isolated wild type yeast mitochondria show rather a network of large lamellae, it makes the hypothesis of tubulation unlikely in yeast. Strauss et al. (2008) proposed that the dimer ribbons of ATP synthase at the apex of cristae enforce a strong local curvature on the membrane due to the angle between the F0 domains, and that they consequently shape the inner mitochondrial membrane. Therefore, the double rows of ATP synthases at the apex of cristae could be involved in the budding of the peripheral inner membrane, thus resulting in the formation of lamellar cristae. We also hypothesize that the absence of ribbon ATP synthase dimers at the apex of cristae of mutants causes the fusion of lamellar cristae, thus leading to long cristae that are characteristic of onion-like structures (Fig. 8). Several roles and functional advantages of dimerization/ oligomerization of ATP synthase have been put forward. Bornhövd et al. (2006) showed that the mitochondrial membrane potential is dependent on the oligomeric state of ATP synthases. They proposed a role for the supracomplexes of the F1 F0 -ATP synthase in organizing microdomains within the inner membrane, ensuring the optimal bioenergetic competence of mitochondria. Related to this is the induction of a local curvature of cristae membranes by the intrinsic angle between two associated ATP synthase at the F0 levels. Thus, the position of ATP synthase at the apex of cristae should increase the local pH gradient leading to ATP synthesis under proton-limited conditions (Strauss et al., 2008). Buzhynskyy
Fig. 8. Hypothetical representation showing the fusion of cristae in mitochondria devoid of supramolecular structures for ATP synthases (B). The white balls represent ATP synthases on cristae under the form of a row of dimers in (A) (wild type) and under monomeric form in (B). IMM: inner mitochondrial membrane.
et al. (2007) also demonstrated that ATP synthesis through rotation torque compensation at stator levels stabilizes the interaction within each ATP synthase, and within dimers and oligomers, thus having an impact on cristae morphology. Acknowledgements We thank Dr. Nadine Camougrand for providing a GFP version targeted to yeast mitochondria. We are grateful to Dr. R. Cooke for his contribution to the editing of the manuscript. This work was supported by grants from the CNRS [ACI Biologie cellulaire, molécu-
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
laire et structurale (BCMS)], the Université Victor Segalen, Bordeaux 2, and the Conseil Régional d’Aquitaine. References Allen RD, Schroeder CC, Fok AK. An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J Cell Biol 1989;108:2233–40. Allen RD. Membrane tubulation and proton pumps. Protoplasma 1995;189:1–8. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schägger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J 1998;17:7170–8. Arselin G, Giraud MF, Dautant A, Vaillier J, Brèthes D, Coulary-Salin B, et al. The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane. Eur J Biochem 2003;270:1875–84. Arselin G, Vaillier J, Salin B, Schaeffer J, Giraud MF, Dautant A, et al. The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology. J Biol Chem 2004;279:40392–9. Bornhövd C, Vogel F, Neupert W, Reichert AS. Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes. J Biol Chem 2006;281:13990–8. Bustos DM, Velours J. The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase. J Biol Chem 2005;280:29004–10. Buzhynskyy N, Sens P, Prima V, Sturgis JN, Scheuring S. Rows of ATP synthase dimers in native mitochondrial inner membranes. Biophys J 2007;93:2870–6. Contamine V, Picard M. Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast. Microbiol Mol Biol Rev 2000;64:281–315. Devenish RJ, Prescott M, Rodgers AJ. The structure and function of mitochondrial F1F0-ATP synthases. Int Rev Cell Mol Biol 2008;267:1–58. Dudkina NV, Heinemeyer J, Keegstra W, Boekema EJ, Braun HP. Structure of dimeric ATP synthase from mitochondria: an angular association of monomers induces the strong curvature of the inner membrane. FEBS Lett 2005;579:5769–72. Dudkina NV, Sunderhaus S, Braun HP, Boekema EJ. Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria. FEBS Lett 2006;580:3427–32. Eubel H, Jänsch L, Braun HP. New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II. Plant Physiol 2003;133:274–86. Fronzes R, Weimann T, Vaillier J, Velours J, Brèthes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits. Biochemistry 2006;45:6715–23. Garí E, Piedrafita L, Aldea M, Herrero E. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 1997;13:837–48. Gavin PD, Prescott M, Devenish RJ. F1F0-ATP synthase complex interactions in vivo can occur in the absence of the dimer specific subunit e. J Bioenerg Biomembr 2005;37:55–66.
1789
Gavin PD, Prescott M, Luff SE, Devenish RJ. Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae. J Cell Sci 2004;117:2333–43. Giraud MF, Paumard P, Soubannier V, Vaillier J, Arselin G, Salin B, et al. Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Biochim Biophys Acta 2002;1555:174–80. Goyon V, Fronzes R, Salin B, di-Rago JP, Velours J, Brèthes D. Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology. J Biol Chem 2008;283:9749–58. Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 1996;24:2519–24. Krause F, Reifschneider NH, Goto S, Dencher NA. Active oligomeric ATP synthases in mammalian mitochondria. Biochem Biophys Res Commun 2005;329:583–90. Lau WC, Baker LA, Rubinstein JL. Cryo-EM structure of the yeast ATP synthase. J Mol Biol 2008;382:1256–64. Mannella CA. Structure and dynamics of the mitochondrial inner membrane cristae. Biochim Biophys Acta 2006;1763:542–8. Minauro-Sanmiguel F, Wilkens S, García JJ. Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis. Proc Natl Acad Sci USA 2005;102:12356–8. Paul MF, Velours J, Arselin de Chateaubodeau G, Aigle M, Guerin B. The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex. Eur J Biochem 1989;185: 163–71. Paumard P, Vaillier J, Coulary B, Schaeffer J, Soubannier V, Mueller DM, et al. The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 2002;21:221–30. Rubinstein JL, Walker JE, Henderson R. Structure of the mitochondrial ATP synthase by electron cryomicroscopy. EMBO J 2003;22:6182–92. Soubannier V, Vaillier J, Paumard P, Coulary B, Schaeffer J, Velours J. In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize. J Biol Chem 2002;277:10739–45. Strauss M, Hofhaus G, Schröder RR, Kühlbrandt W. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 2008;27:1154–60. Thomas D, Bron P, Weimann T, Dautant A, Giraud MF, Paumard P, et al. Supramolecular organization of the yeast F1 F0 -ATP synthase. Biol Cell 2008;100:591–601. Velours J, Arselin G. The Saccharomyces cerevisiae ATP synthase. J Bioenerg Biomembr 2000;4:383–90. Weimann T, Vaillier J, Salin B, Velours J. The intermembrane space loop of subunit b (4) is a major determinant of the stability of yeast oligomeric ATP synthases. Biochemistry 2008;47:3556–63. Wittig I, Schägger H. Advantages and limitations of clear-native PAGE. Proteomics 2005;5:4338–46. Wittig I, Schägger H. Structural organization of mitochondrial ATP synthase. Biochim Biophys Acta 2008;1777:592–8. Wittig I, Velours J, Stuart R, Schägger H. Characterization of domain interfaces in monomeric and dimeric ATP synthase. Mol Cell Proteomics 2008;7:995–1004. Zick M, Rabl R, Reichert AS. Cristae formation-linking ultrastructure and function of mitochondria. Biochim Biophys Acta 2009;1793:5–19.
Contents lists available at ScienceDirect
The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel
Review
Mitochondrial F1 F0 -ATP synthase and organellar internal architecture Jean Velours ∗ , Alain Dautant, Bénédicte Salin, Isabelle Sagot, Daniel Brèthes Université Bordeaux 2, CNRS, Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint Saëns, 33077 Bordeaux Cedex, France
a r t i c l e
i n f o
Article history: Available online 24 January 2009 Keywords: Yeast Mitochondria ATP synthase Supramolecular species
a b s t r a c t The mitochondrial F1 F0 -ATP synthase adopts supramolecular structures. The interaction domains between monomers involve components belonging to the F0 domains. In Saccharomyces cerevisiae, alteration of these components destabilizes the oligomeric structures, leading concomitantly to the appearance of monomeric species of ATP synthase and anomalous mitochondrial morphologies in the form of onion-like structures. The mitochondrial ultrastructure at the cristae level is thus modified. Electron microscopy on cross-sections of wild type mitochondria display many short cristae with narrowed intra-cristae space, whereas yeast mutants defected in supramolecular ATP synthases assembly present a low number of large lamellar cristae of constant thickness and traversing the whole organelle. The growth of these internal structures leads finally to mitochondria with sphere-like structures with a mean diameter of 1 m that are easily identified by epifluorescence microscopy. As a result, ATP synthase is an actor of the mitochondrial ultrastructure in yeast. This paper reviews the ATP synthase components whose modifications lead to anomalous mitochondrial morphology and also provides a schema showing the formation of the so-called onion-like structures. © 2009 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Yeast strains and plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Biochemical procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Fluorescence microscopy analyses of yeast cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Ultrastructural studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. ATP synthase is an actor of mitochondrial ultrastructure in yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. The formation of mitochondrial onion-like structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction The yeast mitochondrial F1 F0 -ATP synthase is a large 600 kDa complex that contains at least 17 distinct subunits (Velours and Arselin, 2000) organized into a catalytic part called F1 and a base piece called F0 . F0 is embedded in the mitochondrial membrane and is mainly composed of hydrophobic subunits forming a specific proton channel. The enzyme uses the proton electrochemical
Abbreviations: BN-PAGE, blue native polyacrylamide slab gel electrophoresis; F1 and F0 , peripheral and integral membrane portions of ATP synthase. ∗ Corresponding author. Tel.: +33 556999001; fax: +33 556999010. E-mail address: [email protected] (J. Velours). 1357-2725/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2009.01.011
1783 1784 1784 1784 1784 1784 1784 1784 1786 1788 1789
gradient generated by the respiratory chain to produce ATP from ADP and inorganic phosphate. This enzyme is a molecular rotary motor where the proton translocation through F0 induces the rotation of a ring of 10 hydrophobic subunits (subunit 9 in yeast). This drives the rotation of a central stalk inside the catalytic head that emerges from the membrane (Devenish et al., 2008 for review). The mitochondrial ATP synthase adopts supramolecular structures that have been found in a large range of organisms by native gel electrophoresis, mainly in dimeric forms (Arnold et al., 1998; Eubel et al., 2003; Krause et al., 2005) but also in higher molecular forms (tetramers, hexamers) according to the electrophoresis technique used (Paumard et al., 2002; Giraud et al., 2002; Krause et al., 2005; Wittig et al., 2008). Depending on the detergent utilized during the extraction and the purification procedures, the
1784
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
isolated complexes were monomers and dimers as shown by electron microscopy single particle analysis (Rubinstein et al., 2003; Lau et al., 2008; Dudkina et al., 2005, 2006; Minauro-Sanmiguel et al., 2005; Thomas et al., 2008), although higher structures such as tetramers have also been reported (Thomas et al., 2008, supplementary data). The isolated ATP synthase dimers adopt a V-like structure with the two F0 parts linked together. Several F0 subunits are essential for dimerization. In Saccharomyces cerevisiae, it has been shown that subunits e and g that are not involved in the ATPase or ATP synthase activity are essential actors for dimerization (Arnold et al., 1998) and oligomerization (Fronzes et al., 2006). These two proteins are present only in mitochondria. Other components such as subunit 4, the homologous subunit to the bsubunit of beef ATP synthase, and other proteins of the peripheral stalk were found to be involved in vivo in the interactions between monomers (Gavin et al., 2005; Fronzes et al., 2006; Wittig and Schägger, 2008). Supramolecular structures of mitochondrial ATP synthase (tetramers, hexamers) have been reported both in blue native gel experiments and in cross-link experiments showing that subunits e and g are involved in dimer stabilization (Fronzes et al., 2006) and in the oligomerization process (Arselin et al., 2003; Krause et al., 2005; Wittig and Schägger, 2005; Bustos and Velours, 2005). Such assemblies have been observed in situ by atomic force microscopy (Buzhynskyy et al., 2007), by transmission electron microscopy (Thomas et al., 2008) and by electron cryo-tomography (Strauss et al., 2008). Originally discovered in Paramecium multimicronucleatum mitochondria (Allen et al., 1989), the supramolecular organization of ATP synthases associates the linear and regular arrays of dimer assembly on cristae to the formation of tubular cristae (Allen, 1995). Surprisingly, yeast mutant mitochondria devoid of either subunit e or g display numerous digitations and onion-like structures, thus suggesting a relationship between ATP synthase dimerization/oligomerization and cristae morphology (Paumard et al., 2002; Soubannier et al., 2002), in agreement with the hypothesis of Allen (1995). This observation was reinforced by the aberrant mitochondrial morphology in cells whose ATP synthase complexes were cross-linked in vivo through subunit ␥-27-DsRed fusion proteins (Gavin et al., 2004). Thus it appears that by promoting supramolecular structures, ATP synthase is one of the numerous actors involved in the establishment of the internal mitochondrial morphology (Mannella, 2006; Zick et al., 2009 for reviews). Here we summarize the alterations in ATP synthase subunits of yeast that lead to mitochondria showing onion-like structures and provide data describing the formation of such objects. 2. Methods 2.1. Yeast strains and plasmids The Saccharomyces cerevisiae strain D273-10B/A/H/U (Mat ␣, met6, ura3, his3) was the wild type strain (Paul et al., 1989). The TIM11 and ATP20 strains were constructed by a PCR-based mutagenesis (Güldener et al., 1996). The strains containing modified versions of subunits e and g were obtained by integration of the mutated versions of TIM11 and ATP20 genes at the chromosomic locus in the respective deleted-disrupted strains (Arselin et al., 2004; Bustos and Velours, 2005). The tetO-TIM11 and tetO-ATP20 strains were constructed as described in Arselin et al. (2004). 2.2. Biochemical procedures Cells were grown aerobically at 28 ◦ C in a complete liquid medium containing 2% lactate as carbon source (respiratory medium) or 2% galactose as indicated. The rho- cell production in
cultures was measured on glycerol plates supplemented with 0.1% glucose in the presence or not of 20 g/ml of doxycycline. Electrophoretic and western blot analyses of mitochondrial proteins are described in Arselin et al. (2004). 2.3. Fluorescence microscopy analyses of yeast cells For visualization of mitochondria by fluorescence microscopy, a pRS303 vector bearing the wild type ATP20 gene under the dependence of a tetracycline-regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase was integrated in the his3 gene of cells devoid of subunit g. The resulting strain was grown for 6 h with 2% galactose as carbon source with or without 20 g/ml of doxycycline. The cells were fixed by formaldehyde addition (3.7% final) directly in the culture. The cells were observed in a fully automated Zeiss 200 M inverted microscope (Carl Zeiss, Thornwood, NY) equipped with an MS-2000 stage (Applied Scientific Instrumentation, Eugene, OR), a Lambda DG4 LS 175-watt xenon light source (Sutter, Novato, CA), a 100 × 1.4 numerical aperture plan-apochromat objective. The filter cube used was an Endow GFP long pass (excitation: HQ470/40, emission: HQ500lp, and beam splitter: Q495lp–Chroma Technology Corp. Rockingham, VT). Images were acquired using a CoolSnap HQ camera (Roper Scientific, Tucson, AZ). The microscope, camera, and shutters (Uniblitz, Rochester, NY) were controlled by SlideBook software (Intelligent Imaging Innovations Inc., Denver, CO). All the pictures are maximum projection of z-stacks acquired with a Z step of 0.2 m. All the image measurements were done using the SlideBook 4.1. software (Intelligent Imaging Innovations Inc., Denver, CO). 2.4. Ultrastructural studies Freezing and freeze substitutions of yeast cells pellets were performed as described in Paumard et al. (2002). 3. Results and discussion 3.1. ATP synthase is an actor of mitochondrial ultrastructure in yeast Our laboratory has shown by electron microscopy that the loss of supramolecular structures of ATP synthases leads to anomalous mitochondria. This anomaly was found in the null mutant in TIM11 and ATP20 genes encoding for subunits e and g, respectively (Paumard et al., 2002), two components associated with ATP synthase and that are involved in the dimerization of the enzyme (Arnold et al., 1998) but not in its activity. The main observation obtained by transmission electron microscopy was the presence of mitochondria sometimes having sphere-like structures with a diameter of 0.8–1 m. These objects are composed of several dense layers separated by white spaces (Paumard et al., 2002). By immune-electron microscopy, the porin protein, which is a standard outer membrane indicator, was found to be restricted to the outer-most membrane, whereas ATP synthase was associated with the inner membranes. Thus, the dense layers correspond to the matrix space whereas the white spaces correspond to the inner membrane space. The abnormal mitochondrial structure was interpreted as the consequence of an uncontrolled biogenesis of the inner mitochondrial membrane. The alterations described above were observed in mutant cells grown for at least 30 generations after deletion of the genes encoding subunits e or g. However, 40% of TIM11 and ATP20 mutant cells spontaneously converted to rho- cells during cultures (Fig. 1), whose effect was to increase the doubling time with nonfermentable medium and to decrease oligomycin sensitivity of ATP synthase. Rho- cells that are altered in their mitochondrial DNA
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
1785
Fig. 1. Appearance of rho- cells upon depletion of subunits e or g. The amount of subunits e and g was modulated as a function of time by using a doxycycline-regulated expression system. Aliquots of cultures were plated to measure the amount of rhocells that were spontaneously generated. e and g refer to strains whose nuclear genes TIM11 and ATP20 encoding the subunits e and g, respectively were inactivated. (1) (tetO-TIM11 cells), (2) (tetO-ATP20 cells). wt, wild type.
have no F0 domain and therefore contain free F1 in the matrix with ATPase activity insensitive to oligomycin. Such a conversion in rhocells was always observed upon modification of genes encoding subunits of yeast ATP synthase (Contamine and Picard, 2000). As
Fig. 3. The lack of ATP synthase subunits e and g leads to an anomalous mitochondrial structure. (A and B) Transmission electron microscopy of tetO-TIM11 cells grown in a respiratory medium without (A) or with doxycycline (B) for 12 h. m = mitochondria. The bar indicates 500 nm.
Fig. 2. The decrease in the amount of subunit e leads to the lack of subunit g and a concomitant decrease in the amount of oligomeric species of yeast ATP synthase. (A and B) Western blot analyses of mitochondria isolated from tetO-TIM11 cells grown in a respiratory medium in the presence of 20 g/ml of doxycycline for 2 and 6 h. The blots were probed with polyclonal antibodies raised against either subunits e and i (A) or subunits g and i (B). (C) BN-PAGE analysis of digitonin extracts of mitochondria isolated from yeast cells grown in the presence of doxycycline for 2, 6 and 12 h. The slab gel was stained with Coomassie brilliant blue. wt = wild type, dox. = doxycycline, oli., dim., and mon. = oligomeric, dimeric and monomeric species of yeast ATP synthase, respectively. The star indicates the location of the (III + IV)2 supercomplex.
a result, it was important to differentiate the effect of the lack of subunits e or g on mitochondrial morphology from that due to spontaneous conversion in rho- cells. To this end, the expression of the TIM11 and ATP20 genes was modulated by using a tetracyclineregulatable promoter system (Garí et al., 1997; Arselin et al., 2004). During the time course of the depletion in either subunit e or g in tetO-TIM11 or tetO-ATP20 cells, the amount of rho- cells remained comparable to that of wild type cells. A small increase in the rho- cell content was noted only after 20 h of culture in the presence of doxycycline (Fig. 1). As a result, the mutant strains were grown for up to 12 h in the presence of the antibiotic during the following experiments. Doxycycline addition to the culture medium of tetO-TIM11 cells led to a rapid decrease in amounts of subunit e and subunit g (Fig. 2A and B). Indeed, it was previously reported that the lack of subunit e induces a loss of subunit g (Arnold et al., 1998). The loss of the two subunits led to the disappearance of the supramolecular forms of the ATP synthase in digitonin extracts (Fig. 2C), which correlates with the change in mitochondrial morphology (Fig. 3), as shown by the presence of onion-like structures already described in the null mutant in genes encoding subunits e and g. The behavior of the mitochondrial network in such mutants was also examined. To this end, a pRS303 vector bearing the wild type ATP20 gene under the dependence of a tetracycline-
1786
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
Table 1 Yeast mutants altered both in ATP synthase ability to promote supramolecular structures and in mitochondrial morphology. Strains
Modification
References
TIM11 ATP20
Disruption of TIM11 gene Disruption of ATP20 gene
Paumard et al. (2002) Paumard et al. (2002)
eG19L e19A eG15L
Alteration of GxxxG motif of subunit e
Arselin et al. (2003)
eK32 stop gV100 stop gR106 stop
Deletion of C-terminal part of subunit e Deletion of C-terminal part of subunit g Deletion of C-terminal part of subunit g
Unpublished data Bustos and Velours (2005) Bustos and Velours (2005)
gG101L gG105L g102A
Alteration of GxxxG motif of subunit g
Bustos and Velours (2005)
4TM1 4(49–55) RFY5-1 tetO-TIM11 tetO-ATP20 ␥-27-DsRed
Deletion of first 43 amino acid residues of subunit 4 Deletion in loop 49–55 of subunit 4 Controlled depletion of subunit h Controlled depletion of subunit e Controlled depletion of subunit g DsRed attached to the C-terminus of subunit ␥
Soubannier et al. (2002) Weimann et al. (2008) Goyon et al. (2008) Arselin et al. (2004) Arselin et al. (2004) Gavin et al. (2004)
regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase was integrated in the his3 gene of cells devoid of subunit g. Cells were grown for 6 h with 2% galactose as carbon source with or without doxycycline and observed by epifluorescence microscopy. In the absence of doxycycline, a typical elongated and branched mitochondrial network was present (Fig. 4). After 6 h of growth in the presence of the antibiotic, the tubular mitochondrial network changed to punctuated roundedshape organelles. Indeed a careful analysis along the z-axis showed that these objects are hollow spheres probably corresponding to the mitochondria with onion-like structures found by transmission electron microscopy. The mean diameter of these objects is 1.05 ± 0.4 m (175 objects measured) and their mean number per cell is 2.43 ± 1.89 (72 cells were scored). The size of these objects is in agreement with that observed by transmission electron microscopy. These data clearly indicate that the yeast mitochondrial network is strongly altered in the absence of subunit g. As a consequence, the lack of supramolecular structures of ATP synthase might be considered as one of the parameters influencing mitochondrial morphology. Since the first experiments where subunit e or g was removed from the enzyme, new data have reinforced the link between the ability of ATP synthase to dimerize and the altered morphology.
Table 1 summarizes alterations in ATP synthase subunits that lead both to the absence of ATP synthase dimerization and altered mitochondrial morphology. Subunits e and g display a conserved GxxxG dimerization motif in the hydrophobic part of the sequence. The glycine residues were replaced by leucine to increase the steric hindrance and alternatively an alanine residue was inserted after the first glycine in the GxxxG motif. All these alterations lead to the lack of ATP synthase dimerization and to the presence of mitochondria with onion-like structures (Arselin et al., 2003; Bustos and Velours, 2005). Subunit 4, the homologous protein to the b-subunit, is also involved in the dimerization process. The eukaryotic mitochondrial protein displays a transmembranous domain in its N-terminus composed of two hydrophobic segments. The removal of the first 43 amino acid residues containing the first membrane-spanning segment did not alter the function of ATP synthesis, but led to the lack of subunit g and as a consequence to the loss of supramolecular structures for the ATP synthase and the appearance of onion-like structures (Soubannier et al., 2002). From these data and crosslinking experiments, we hypothesized that in eukaryotic cells the b-subunit has evolved to accommodate the interaction with the gsubunit that is an associated ATP synthase component only present in the mitochondrial enzyme. In addition, the shortening of loop 47–55 linking the two membrane-spanning segments of subunit 4 significantly alters the oligomerization of the yeast ATP synthase and as a consequence the mitochondrial morphology (Weimann et al., 2008). Another question is the ultrastructure of isolated mitochondria from cells showing anomalous mitochondrial morphologies. Like isolated wild mitochondria, mitochondria devoid of either subunit e or g are also circular but display numerous membranes in the matrix space that traverses the organelle (Arselin et al., 2004). Indeed, the aspect of isolated mitochondria is certainly far from reality and probably results from alterations in the ultrastructure of organelles during isolation. 3.2. The formation of mitochondrial onion-like structures
Fig. 4. The absence of ATP synthase subunit g alters the mitochondrial network. The construction of the strain harboring the wild type ATP20 gene under the dependence of a tetracycline-regulatable promoter system and the GFP gene fused to the leader sequence of citrate synthase is described in Section 2. Cells were grown for 6 h in a galactose medium as carbon source in the presence or not of 20 g/ml of doxycycline and examined by epifluorescence microscopy. The bar indicates 2 m.
An open question is the formation of onion-like structures that display successive layers of membranes. Fig. 5 shows wild type yeast mitochondria in the cellular context. With our sample preparation procedure (Paumard et al., 2002), the cristae of the wild type cells are small with membranes joined in such a way that the intra-cristae space is not visible. In Fig. 6A and B, the mitochondrial morphology of the mutant eG15L is shown as an example. The mutation alters the conserved GxxxG domain
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
1787
Fig. 5. Wild type mitochondria have short cristae and a narrow intracristae space. Wild type yeast cells were grown in a respiratory medium and prepared for transmission electron microscopy as in (Paumard et al., 2002). The arrows indicate cristae. The bar indicates 100 nm.
described above and as a consequence prevents ATP synthase dimerization/oligomerization. Transmission electron microscopy of the eG15L mutant exemplifies a typical organellar ultrastructure. The mitochondria appear elongated. Fig. 6A shows mitochondria with unique lamellar cristae whose thickness is evaluated at 30 nm. The two cristae membranes delimit a 12 nm white space that is nearly constant over almost all the organelle except at the cristae junction level, where a thin 3 nm channel is observed. The compartments were identified by immunogold labeling (Paumard et al., 2002). The two dense layers on both sides of the cristae correspond to the matrix space and the white space corresponds to the intracristae compartment. The outer membrane lies at the periphery of the organelle and is probably joined with the inner peripheral membrane. Other mitochondria display two (Fig. 6B) and sometimes three (not shown here) lamellar cristae. In all the samples, the main point is the presence of lamellar structures traversing the organelle when the ATP synthase lacks its capacity to oligomerize. Indeed, intermediary organelles between “normal” and onion-like mitochondria can exist. An example is given with mutant 4(49–55). In this strain the loop linking the two membrane-spanning segments of subunit 4 was shortened. Despite the presence of subunits e and g, most of ATP synthases showed as monomers on CN-PAGE analysis of digitonin extracts (Weimann et al., 2008). The cristae are frequently pinched in this mutant (arrows in Fig. 6C) and the intermembrane space appears larger than that of wild-type cristae. The formation of onion-like structures was interpreted as an uncontrolled biogenesis of inner membrane sheets, which promotes mitochondrial elongation and leads to large organelles inside the cells. Transmission electron micrographs (Fig. 7) show the formation of onion-like structures in a cell progressively devoid of either subunit e or g. The decrease in the expression of subunit e leads to spherical mitochondria (left of the figure) with several cristae that traverse the mitochondria (corresponding to 4 h of growth with doxycycline). Note the large intra-cristae space that differs significantly from that of wild type mitochondria. The growth of several lamellar sheets of the inner membrane enlarges
Fig. 6. Anomalous structure of mitochondrial cristae in yeast mutants devoid of supramolecular structures of ATP synthase. eG15L mutant (A and B) and 4(49–55) (C). Cells were grown in a respiratory medium and prepared for transmission electron microscopy as in Paumard et al. (2002). The insert enlarges a section of the inner structure of mutant mitochondria. The arrows indicate spots where membranes of cristae are joined. The bar indicates 500 nm.
the organelle and gives the aspect of a saucer and open or closed spherical structures as seen by electron microscopy, depending on the cutting plane of the samples (corresponding to 12 h of growth with doxycycline). The size of the objects is in agreement with that observed by epifluorescence microscopy. The anomalous structures are reversible. Using the doxycycline-regulatable expression system, Arselin et al. (2004) showed that onion-like structures are formed after five generations. When doxycycline is removed from the culture medium, normal cristae are observed after five generations, corresponding to a recovery of both subunits e and g and supramolecular forms of ATP synthase. Several points emerge from these data. In mutants devoid of supramolecular associations of ATP synthase, the ultrastructure of cristae is drastically modified. Whereas wild type cristae are short, mutant cristae traverse the whole organelle longitudinally and the intra-cristae space is drastically increased (12 nm). This indicates that the intra-cristae space is able to move from the closed state observed in wild type mitochondria (Fig. 5) to the relaxed state of onion-like structures (Fig. 6). The constant size of the intra-cristae space in mutant cells suggests the existence of structures inside this space that keep the distance constant between the two cristae membranes. However, these structures are probably lost during
1788
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
Fig. 7. Representation model of the formation of yeast mitochondrial onion-like structures during depletion of subunit e or g (→) and their visualization by electron microscopy according to the cutting plane of the samples (- - ->). wt = wild type. The 3D model of objects was created by using Google Sketchup.
the isolation of mitochondria since the cristae appear larger and swollen except at the cristae junction (Arselin et al., 2004). Electron tomography of isolated yeast wild type mitochondria demonstrated a three-dimensional network of large lamellae that were interconnected in some places (Thomas et al., 2008). In addition, transmission electron microscopy of negatively stained submitochondrial particles revealed the presence of two parallel rows of ATP synthase along the membrane border with a density of 7–10 pairs of particles for 100 nm (Thomas et al., 2008). Similar data were obtained by cryo-electron tomography of bovine heart mitochondria where rows of dimers were found in the most tightly curved membrane regions of cristae (Strauss et al., 2008). According to Allen (1995), ATP synthase oligomerization could be responsible for the tubulation of the mitochondrial inner membrane allowing the formation of cristae, which in fine increases the surface of the inner membrane available for components of oxidative phosphorylation. Since the tomographic analysis of isolated wild type yeast mitochondria show rather a network of large lamellae, it makes the hypothesis of tubulation unlikely in yeast. Strauss et al. (2008) proposed that the dimer ribbons of ATP synthase at the apex of cristae enforce a strong local curvature on the membrane due to the angle between the F0 domains, and that they consequently shape the inner mitochondrial membrane. Therefore, the double rows of ATP synthases at the apex of cristae could be involved in the budding of the peripheral inner membrane, thus resulting in the formation of lamellar cristae. We also hypothesize that the absence of ribbon ATP synthase dimers at the apex of cristae of mutants causes the fusion of lamellar cristae, thus leading to long cristae that are characteristic of onion-like structures (Fig. 8). Several roles and functional advantages of dimerization/ oligomerization of ATP synthase have been put forward. Bornhövd et al. (2006) showed that the mitochondrial membrane potential is dependent on the oligomeric state of ATP synthases. They proposed a role for the supracomplexes of the F1 F0 -ATP synthase in organizing microdomains within the inner membrane, ensuring the optimal bioenergetic competence of mitochondria. Related to this is the induction of a local curvature of cristae membranes by the intrinsic angle between two associated ATP synthase at the F0 levels. Thus, the position of ATP synthase at the apex of cristae should increase the local pH gradient leading to ATP synthesis under proton-limited conditions (Strauss et al., 2008). Buzhynskyy
Fig. 8. Hypothetical representation showing the fusion of cristae in mitochondria devoid of supramolecular structures for ATP synthases (B). The white balls represent ATP synthases on cristae under the form of a row of dimers in (A) (wild type) and under monomeric form in (B). IMM: inner mitochondrial membrane.
et al. (2007) also demonstrated that ATP synthesis through rotation torque compensation at stator levels stabilizes the interaction within each ATP synthase, and within dimers and oligomers, thus having an impact on cristae morphology. Acknowledgements We thank Dr. Nadine Camougrand for providing a GFP version targeted to yeast mitochondria. We are grateful to Dr. R. Cooke for his contribution to the editing of the manuscript. This work was supported by grants from the CNRS [ACI Biologie cellulaire, molécu-
J. Velours et al. / The International Journal of Biochemistry & Cell Biology 41 (2009) 1783–1789
laire et structurale (BCMS)], the Université Victor Segalen, Bordeaux 2, and the Conseil Régional d’Aquitaine. References Allen RD, Schroeder CC, Fok AK. An investigation of mitochondrial inner membranes by rapid-freeze deep-etch techniques. J Cell Biol 1989;108:2233–40. Allen RD. Membrane tubulation and proton pumps. Protoplasma 1995;189:1–8. Arnold I, Pfeiffer K, Neupert W, Stuart RA, Schägger H. Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits. EMBO J 1998;17:7170–8. Arselin G, Giraud MF, Dautant A, Vaillier J, Brèthes D, Coulary-Salin B, et al. The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane. Eur J Biochem 2003;270:1875–84. Arselin G, Vaillier J, Salin B, Schaeffer J, Giraud MF, Dautant A, et al. The modulation in subunits e and g amounts of yeast ATP synthase modifies mitochondrial cristae morphology. J Biol Chem 2004;279:40392–9. Bornhövd C, Vogel F, Neupert W, Reichert AS. Mitochondrial membrane potential is dependent on the oligomeric state of F1F0-ATP synthase supracomplexes. J Biol Chem 2006;281:13990–8. Bustos DM, Velours J. The modification of the conserved GXXXG motif of the membrane-spanning segment of subunit g destabilizes the supramolecular species of yeast ATP synthase. J Biol Chem 2005;280:29004–10. Buzhynskyy N, Sens P, Prima V, Sturgis JN, Scheuring S. Rows of ATP synthase dimers in native mitochondrial inner membranes. Biophys J 2007;93:2870–6. Contamine V, Picard M. Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast. Microbiol Mol Biol Rev 2000;64:281–315. Devenish RJ, Prescott M, Rodgers AJ. The structure and function of mitochondrial F1F0-ATP synthases. Int Rev Cell Mol Biol 2008;267:1–58. Dudkina NV, Heinemeyer J, Keegstra W, Boekema EJ, Braun HP. Structure of dimeric ATP synthase from mitochondria: an angular association of monomers induces the strong curvature of the inner membrane. FEBS Lett 2005;579:5769–72. Dudkina NV, Sunderhaus S, Braun HP, Boekema EJ. Characterization of dimeric ATP synthase and cristae membrane ultrastructure from Saccharomyces and Polytomella mitochondria. FEBS Lett 2006;580:3427–32. Eubel H, Jänsch L, Braun HP. New insights into the respiratory chain of plant mitochondria. Supercomplexes and a unique composition of complex II. Plant Physiol 2003;133:274–86. Fronzes R, Weimann T, Vaillier J, Velours J, Brèthes D. The peripheral stalk participates in the yeast ATP synthase dimerization independently of e and g subunits. Biochemistry 2006;45:6715–23. Garí E, Piedrafita L, Aldea M, Herrero E. A set of vectors with a tetracycline-regulatable promoter system for modulated gene expression in Saccharomyces cerevisiae. Yeast 1997;13:837–48. Gavin PD, Prescott M, Devenish RJ. F1F0-ATP synthase complex interactions in vivo can occur in the absence of the dimer specific subunit e. J Bioenerg Biomembr 2005;37:55–66.
1789
Gavin PD, Prescott M, Luff SE, Devenish RJ. Cross-linking ATP synthase complexes in vivo eliminates mitochondrial cristae. J Cell Sci 2004;117:2333–43. Giraud MF, Paumard P, Soubannier V, Vaillier J, Arselin G, Salin B, et al. Is there a relationship between the supramolecular organization of the mitochondrial ATP synthase and the formation of cristae? Biochim Biophys Acta 2002;1555:174–80. Goyon V, Fronzes R, Salin B, di-Rago JP, Velours J, Brèthes D. Yeast cells depleted in Atp14p fail to assemble Atp6p within the ATP synthase and exhibit altered mitochondrial cristae morphology. J Biol Chem 2008;283:9749–58. Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res 1996;24:2519–24. Krause F, Reifschneider NH, Goto S, Dencher NA. Active oligomeric ATP synthases in mammalian mitochondria. Biochem Biophys Res Commun 2005;329:583–90. Lau WC, Baker LA, Rubinstein JL. Cryo-EM structure of the yeast ATP synthase. J Mol Biol 2008;382:1256–64. Mannella CA. Structure and dynamics of the mitochondrial inner membrane cristae. Biochim Biophys Acta 2006;1763:542–8. Minauro-Sanmiguel F, Wilkens S, García JJ. Structure of dimeric mitochondrial ATP synthase: novel F0 bridging features and the structural basis of mitochondrial cristae biogenesis. Proc Natl Acad Sci USA 2005;102:12356–8. Paul MF, Velours J, Arselin de Chateaubodeau G, Aigle M, Guerin B. The role of subunit 4, a nuclear-encoded protein of the F0 sector of yeast mitochondrial ATP synthase, in the assembly of the whole complex. Eur J Biochem 1989;185: 163–71. Paumard P, Vaillier J, Coulary B, Schaeffer J, Soubannier V, Mueller DM, et al. The ATP synthase is involved in generating mitochondrial cristae morphology. EMBO J 2002;21:221–30. Rubinstein JL, Walker JE, Henderson R. Structure of the mitochondrial ATP synthase by electron cryomicroscopy. EMBO J 2003;22:6182–92. Soubannier V, Vaillier J, Paumard P, Coulary B, Schaeffer J, Velours J. In the absence of the first membrane-spanning segment of subunit 4(b), the yeast ATP synthase is functional but does not dimerize or oligomerize. J Biol Chem 2002;277:10739–45. Strauss M, Hofhaus G, Schröder RR, Kühlbrandt W. Dimer ribbons of ATP synthase shape the inner mitochondrial membrane. EMBO J 2008;27:1154–60. Thomas D, Bron P, Weimann T, Dautant A, Giraud MF, Paumard P, et al. Supramolecular organization of the yeast F1 F0 -ATP synthase. Biol Cell 2008;100:591–601. Velours J, Arselin G. The Saccharomyces cerevisiae ATP synthase. J Bioenerg Biomembr 2000;4:383–90. Weimann T, Vaillier J, Salin B, Velours J. The intermembrane space loop of subunit b (4) is a major determinant of the stability of yeast oligomeric ATP synthases. Biochemistry 2008;47:3556–63. Wittig I, Schägger H. Advantages and limitations of clear-native PAGE. Proteomics 2005;5:4338–46. Wittig I, Schägger H. Structural organization of mitochondrial ATP synthase. Biochim Biophys Acta 2008;1777:592–8. Wittig I, Velours J, Stuart R, Schägger H. Characterization of domain interfaces in monomeric and dimeric ATP synthase. Mol Cell Proteomics 2008;7:995–1004. Zick M, Rabl R, Reichert AS. Cristae formation-linking ultrastructure and function of mitochondria. Biochim Biophys Acta 2009;1793:5–19.