the f1fo - atpsynthase is an h - pumping atpase evolutionary specialized in synthesizing atp by using an h gradient generated across a biological membrane . it is present in bacteria and intracellular organelles such as chloroplasts and mitochondria . in these , the enzyme is hosted within the inner membrane as part of the oxphos , where it couples the transport of h from the intermembrane space into the matrix with the synthesis of atp , guaranteeing the supply of energy to biological processes , since the majority of cellular atp is generated by the mitochondrial f1fo - atpsynthase . the molecular structure , catalytic mechanism , and regulation of the mitochondrial f1fo - atpsynthase were described by the seminal work of the nobel laureates mitchell , boyer and walker , that revealed its complexity and the functional steps that drive the synthesis of atp . besides its role as energy producer , the mitochondrial f1fo - atpsynthase is also essential for the maintenance of the mitochondrial membrane potential ( m ) which is crucial for the import of proteins into the organelle and cristae structure . in addition , it is fundamental for an optimal supramolecular organization of the respiratory chain , and for regulating the mobilization of cytochrome c during apoptosis . in animals and plants , the mitochondrial f1fo - atpsynthase is molecularly regulated by an endogenous , nuclear - encoded polypeptide , the atpase inhibitory factor 1 ( if1 ) . if1 is primarily responsible for inhibiting the hydrolysis of atp by the atp synthase , an event that occurs when the electrochemical proton gradient across the mitochondrial inner membrane is lost ( e.g. , during hypoxic / ischaemic conditions ) , and the enzyme reverses to restore m . a number of studies have shown that if1 is also involved in the regulation of the oligomeric state of the f1fo - atpsynthase , by facilitating the enzyme 's dimerization via a molecular link between two f1 domains ; for this reason , it is also implicated in the remodelling of cristae structure , and consequently in the regulation of mito - ultrastructure and morphology . the interest for this inhibitor , or regulator  as we like to consider it  stems from many reasons ; among them , the evidence for a key role in pathologies is the most meaningful but the less explored . if1 overexpression is reported in human carcinomas , differences in the ratio of expression between if1 and the f1fo - atpsynthase are related to changes in cellular responses to ischaemia / reperfusion injury [ 11 , 12 ] , and its absence is recorded in a rare form of mitochondrial myopathy called luft 's disease [ 13 , 14 ] . despite this , if1 seems underconsidered in pathologies whose etiology correlates with defective mitochondrial f1fo - atpsynthase . here , we will explain why the interaction between if1 and f1fo - atpsynthase is important , and why the quality of cellular bioenergetics depends on it . it is a multisubunit complex ( ~5,000 aminoacid residues , with a mass of ~600  kda ) consisting of an intrinsic membrane domain , fo ( ~1,500 aminoacids ) , and a globular catalytic domain , f1 ( ~3,500 aminoacids ) , which extends into the mitochondrial matrix ( figure 1 ) . in mammals , the enzyme contains 15 different subunits , nine of which form the fo domain ( a , a6l , b , c , d , f , f6 , e , and g ) , while the f1 domain is instead composed of only six (  ,  ,  ,  ,  , and oscp ) [ 1517 ] . the inhibitory factor 1 ( if1 ) is often regarded as the 16th subunit , although we will learn in this review that the protein is far more correctly defined as its endogenous regulator .   the enzyme could be ideally divided into 4 principal subdomains : the catalytic headpiece ( 33 ) , hosting the three catalytic sites for atp synthesis ( one in each -subunit ) , the h channel ( ac815 ) and two stalks , the central rotor (  ) and the peripheral stator ( bd(f6)oscp ) , that link the first two subdomains together . the remaining minor subunits e , f , g , and a6l are associated with the proton channel ; in particular , subunits e and g are involved in the dimerization of the complex   ( a scheme of the structure of the atp synthase is reported in figure 1 ) . the crystal structure of the mitochondrial f1-atpsynthase , extracted from bovine heart mitochondria , was revealed at the beginning of the nineties [ 19 , 20 ] . in this structure , f1 appears like a flattened sphere of 80   in height and 100   in width , with three - and three -subunits arranged alternately forming a cylinder around the coiled - coil structure of the -subunit . sequences of - and -subunits are 20% identical , and this homology is reflected in their similar folds . all -subunits have almost the same conformation , while the three -subunits adopt three different tertiary structures and are in three diverse nucleotide - bound states : the first , named tp , hosts the binding site for atp ; the second , called dp , binds with high - affinity adp and pi ; the third , e , does not efficiently bind any nucleotide . this asymmetry in the conformation and nucleotide occupancy of -subunits supports the binding change mechanism of catalysis theorized by boyer in the early 1970s , and fully developed in 1993 [ 21 , 22 ] . according to this model , the three catalytic sites can be in three different conformations at any given time . tp and dp have , respectively , a  tight  ( t ) and a  loose ( l ) form , in which adp and pi are converted into atp , while the  open  conformation ( o ) , adopted by e , permits the release of the newly formed atp and the acceptance of another molecule of adp . adp + pi are theorized to enter into an o -subunit , which then assumes an l conformation allowing the synthesis of atp . after the synthesis , subunit  is in the t conformation , before assuming the o conformation in order to release atp . the sequential interconversion between these different conformations , driven by the rotation of the central -subunit relative to the ( )3 subcomplex , enables the catalysis . unfortunately , there is less structural information on the fo domain of atp synthase than the f1 complex . in the ring , subunits c , whose structure was determined by both nmr spectroscopy [ 23 , 24 ] and x - ray crystallography , folds as a hairpin composed by two transmembrane -helices linked by a polar loop ; part of the ring is in contact with subunit a , formed presumably by five transmembrane -helices and containing two proton half - channels that do not span the membrane . to briefly describe the functioning of the f1fo - atpsynthase and its rotational catalysis , we must divide it into two parts : a central  rotor  ( f1-foc10 ) and a surrounding  stator  ( f133-foab2 ) . the driving force for the rotation is generated by the electron - transport chain ( etc ) and is based on the magnitude of the proton electrochemical potential across the inner membrane ; this gradient allows h to flow through the fo domain ( the c - ring ) causing the rotation of the whole  rotor . the precession of the -subunit , which contacts only one -subunit at a time , induces cyclic changes in the  stator  so that atp can be synthesized . the rotational mechanism is extremely complex and depends on the structures of subunits a and c ; the latter contain , in the middle of their c - terminal -helix , a critical aminoacid residue , d61 , which can either be in a protonated ( lipophilic ) or unprotonated ( hydrophilic ) form . protonation and deprotonation of this aspartic acid residue is at the basis of the c - ring rotation dictating the membrane affinity of the single subunits . this occurs only in an aqueous environment and is realized when subunit c is in contact with subunit a , and the aspartate residue is hosted in one of the proton half - channels ( see   for further details ) . in respiring mitochondria , the h motive force ensures the entrance of h residing in the intermembrane space into the cytosolic half - channel . the [ h ] is more than 25 times higher on the cytosolic side than on the matrix side , and the m of 140180  mv increases the [ h ] near the mouth of the cytosolic half - channel , thus resulting in protonation of the enclosed aspartate . once the key residue is protonated , the c - ring can rotate clockwise ( looking from the intermembrane space side ) , and the d61 of a new subunit c enters the matrix half - channel of subunit a , releasing a h into the mitochondrial matrix . thus , the difference in [ h ] and potential on the two sides of the membrane leads to different probabilities of protonation through the two half - channels , which yields directional rotational motion . the coupling between the rotation of the c - ring and the conformational changes in the ( )3 barrel is guaranteed by the tight link of the ring with the central stalk ; while the c - ring rotates , the -subunit turns inside the 33-hexamer , whose rotation is blocked by the presence of the peripheral stalk . thus , the proton - gradient - driven rotation of the c - ring drives the rotation of the -subunit , which in turn promotes the synthesis of atp through the binding - change mechanism . a highly conserved acidic cluster sequence in the c - terminal helical domain of the -subunit ( the delseed motif ) is thought to be essential for atp synthesis by coupling catalysis and rotation . after the binding of an adenosine nucleotide in the catalytic site , the c - terminus is lifted up to the nearly immobile n - terminal part of the protein , and the -delseed sequence is moved in contact with the -subunit , probably allowing the coupling of the -subunit torque with the rotation of the f1 domain ( this hypothesis is still controversial ) . each 360 rotation of the -subunit leads to the synthesis and release of three molecules of atp . the number of subunits in the c - ring , which ranges from 8 ( in bovine and probably in all vertebrates and invertebrates fo domain ) to 15 ( in spirulina platensis ) , determines the number of protons that are required to generate a molecule of atp . mitochondria are the sites where cellular energy is most abundantly produced , due to the constant activity of the mitochondrial f1fo - atpsynthase . although publications have suggested that it may also be localized on the plasma membrane [ 32 , 33 ] , we shall be exclusively discussing and referring to that embedded in the mitochondrial inner membrane . as the f1fo - atpsynthase is a reversible nanomotor , it can also hydrolyze atp by translocating h from the matrix into the intermembrane space ( an event that vigorously occurs when the enzyme is in isolation ) . it does so to maintain the mitochondrial membrane potential ( m ) at a suboptimal level during deenergized conditions that occur when respiration is impaired by defects in the activity of the etc , or when the mitochondrial inner membrane is leaky due to alterations in its structural integrity . the m is not only important for atp production , but also for mitochondrial protein import and assembly . disruption of the m is therefore implicated in various apoptotic phenomena ( see ) , being its maintenance crucial for cell viability . the reversal of the f1fo - atpsynthase is avoidable in eukaryotes and the enzyme must be controlled to prevent futile hydrolysis of atp when the transmembrane proton electrochemical gradient collapses . only facultative anaerobic bacteria employ this method for generating a vital proton electrochemical gradient in the absence of oxygen . when reversal of the f1fo - atpsynthase does occur , the depletion of cellular atp can be more or less severe depending on the energy requests of the tissue , but in organs with high atp demand , like brain or skeletal muscle , or in case of augmented atp request , cellular demise is tangibly accelerated . apart from pathological states , repression or upregulation of atp synthesis normally occurs in physiological conditions when intracellular atp levels are , respectively , sufficiently high or too low . it has been calculated that , in eukaryotic cells , the rate of atp utilization changes by a factor of 510 , such as during exercise and/or acclimatization . several are the mechanisms by which the activity of the f1fo - atpsynthase is regulated : ( a ) transcriptional factors [ 39 , 40 ] ; ( b ) translational control [ 4143 ] ; ( c ) modulation of the electron transport chain or the citric acid cycle [ 44 , 45 ] ; ( d ) adp inhibition   and ( e ) regulatory proteins , such as if1 . although recent evidence has also suggested that the oncoprotein bcl - xl interacts with the f1fo - atpsynthase [ 46 , 47 ] , if1 is the only molecular regulator of the enzyme characterized both biochemically and functionally . nonetheless , other proteins which directly interact with the enzyme have been identified , such as factor b , essential for atp synthesis and implicated in the regulation of the f1fo - atpsynthase oligomerization , cabi , which may up - regulate the enzyme in response to increased cytoplasmic ca , and finally s100a1 , which has been found to enhance the enzymatic performance in cardiac muscle . if1 was discovered in 1963 by pullman and monroy   in mitochondria from bovine hearts ( a schematic representation of the structure of bovine if1 is reported in figure 2(a ) ) . to date , if1 homologues have been isolated from other mammals ( e.g. , rat   and human ) , yeast ( saccharomyces cerevisiae   and candida utilis ) and plants . if1 is a small , basic , heat - stable protein of approximately 10  kda ( in mammals the mature form of the polypeptide is composed of a number of amino acid residues which ranges from 81  in human , chimpanzee , dog and mouse , to 84  in cow ) . it is predominantly compartmentalized inside the mitochondrial matrix ( figure 2(b ) ) , although studies have proposed that if1 is also present in the cytosol and on the plasma membrane , as well as secreted into the extracellular environment , where it is implicated in the modulation of the activity of endothelial cells . intriguingly , in this very extramitochondrial localization , a role for hepatic hdl - cholesterol and triglyceride metabolism was also proposed [ 32 , 57 ] . if1 interacts with the catalytic subunit of the f1fo - atpsynthase , inhibiting the hydrolysis of atp under conditions that favour the reversion of the enzyme activity ( figure 3(b ) ) . the regulatory protein is therefore an indispensable component to protect the cell from atp depletion - driven damage and demise . if1 completely inhibits , through a noncompetitive mechanism , the atp - hydrolyzing activity of the f1fo - atpsynthase without affecting the synthesis of atp during oxidative phosphorylation , although a few studies argue differently on this [ 58 , 59 ] ; nevertheless , if1 is reported to be largely active only at low ph , hence in conditions of atp hydrolysis . the inhibitory protein binds to the soluble f1 domain in a 1  :  1 stoichiometry in the presence of mg and atp . this is important for maintaining cellular atp , by preventing its hydrolysis , when the h electrochemical gradient across the mitochondrial inner membrane is lost ( e.g. , during hypoxic / ischaemic conditions ) and the enzyme reverses its activity to transiently restore m [ 58 , 59 ] . if1 is encoded by the atpif1 gene , localized at chromosome 1 , and is synthesized as a propolypeptide ( 106 residues in humans ) harbouring a highly conservedn - terminal presequence of 25 amino acids ( identified by the comparison between the amino acid sequences deduced from the cdna and the purified protein ) presumably important for the mitochondrial targeting of the protein . the mature polypeptide ( 8184 aa in mammals ) is significantly conserved among various species . the human protein exhibits 6774% of sequence homology with other known mammalian if1 inhibitors , and also partial homology with the yeast inhibitor inh1p . interestingly , there is a strong correlation between the high sequence conservation and function , as if1 from one species is able to inhibit the f1fo - atpsynthase from another , including yeast [ 51 , 62 , 63 ] . conversely , yeast if1 is not able to inhibit the animal f1 domain because its activity is stabilized by accessory proteins which have no homologues in animals . the most conserved segment of the inhibitory factor is the region that interacts with the f1fo - atpsynthase , comprising amino acid residues 2050 ( 90% identity between the human and bovine sequences , 80% between bovine and rodent ) , and a lot of research into understanding the minimal inhibitory sequence of if1 has focused on this sequence . van raaij and coworkers investigated this , by measuring the activity of several truncated forms of the protein . the intact and truncated forms assayed for inhibition of f1fo - atpsynthase using if1-depleted submitochondrial particles revealed that the minimal inhibitory sequence consists of residues 1447 . in 2001 , ichikawa and colleagues   showed by amino acid replacement that in yeast five residues ( f17 , r20 , r22 , e25 and f28 ) are essential for the inhibitory activity of the protein . two years later it was brought up that six homologous residues of bovine if1 ( f22 , r25 , e26 , q27 , e30 , and y33 ) , which form a cluster on the surface of the -helix , represent the inhibitory site of the protein . the molecular structure of if1 was initially characterized by cabezon et al . in 2001 the protein is -helical along most of its length ( ~90   ) and is active as a dimer at low ph ( < 6.7 ) . the dimerization of two if1 monomers involves their c - terminal regions ( residues 3784 ) , which form an antiparallel double - stranded coiled - coil unit stabilized by complimentary hydrophobic interactions between the two helices , involving residues 4981 . within the dimer , the two minimal inhibitory sequences are at the opposite ends ( the dimer shows an end - end distance of at least 130   , while the distance between the two inhibitory regions is 62   ) and can react with two f1 domains simultaneously ( figure 3(c ) ) . when the ph is above neutrality ( 7.0 - 8.0 ) , the dimers can assemble inactive tetramers or higher oligomers by forming antiparallel coiled - coils in the n - terminal regions ( residues 3244 are involved ) . every protomer of the dimer can participate in two coiled - coil units with two different helices , binding two dimers simultaneously . the formation of the oligomers masks the inhibitory sequences of the dimers , so that if1 can not bind the f1fo - atpsynthase ( figure 3(a ) ) . mammalian if1 contains five highly conserved histidines ( at positions 48 , 49 , 55 , 56 and 70 ) that , if chemically modified or replaced , lead to a complete loss of the ph - susceptible activity of the protein without affecting its inhibitory capacity [ 60 , 71 ] . this histidine - rich region ( residues 4870 ) is involved in the ph sensing mechanism of bovine if1 , and undergoes conformational changes depending on acidity or alkalinity of the environment . critical for the ph - dependent interconversion between the two aggregation states of the polypeptide is the histidine 49 [ 70 , 71 ] . it was observed that replacement of this residue with a different amino acid induces full activation of if1 at ph 8 , and abolishes the ability of the dimers to form oligomers [ 67 , 71 ] . the five histidines seem to be important for the ph - regulated decrease in activity between ph 6.7 and 8.0 , even if they may not represent the only mechanism responsible for such regulation . in fact , a ph - dependent activity was also observed in the if1 2246 peptide   and detected in a 12-residue segment from 32 to 43 ; moreover , h49 is not conserved in yeast , suggesting a diverse ph - sensitivity of the protein . apart from controlling the oligomerization of the polypeptide and consequently the availability of its inhibitory site , the ph itself was proposed to represent the switch between inactive and active forms by controlling the helical content and the flexibility of the whole protein . at low ph ( ~6.7 ) , the helical content seems to be lower , so that the n - terminal region is less ordered and , instead of forming a coiled - coil unit with two other dimers , assumes the correct conformation for binding the f1 complex . however , this theory is controversial since ph was thought to simply act by modulating the electrostatic interactions between the polar residue - enriched n - terminal regions of the dimers , as presented by cabezon et al . . recently , ando and ichikawa   discovered that ph could effectively change the conformation of the active site by acting on a highly conserved glutamate residue , e26 in bovine if1 or e21 in yeast if1 ( h49 is not conserved in yeast and , as a consequence , can not represent the only ph sensor residue of the protein ) . the mechanism of ph - dependency mediated by glutamate regulates only the inhibitory activity of the f1-binding site and not the aggregation state of the polypeptide . very little is known about the transcriptional and posttranscriptional regulations of if1 , despite speculations on a possible contribution by the hypoxia - inducible factor 1- ( hif1- )   and evidence for a downregulation mediated by the immediate early response gene x-1 ( iex-1 ) . the molecular crystal structure of bovine if1:f1-atpase complex is available [ 69 , 77 ] . in the complex , if1 shows an ordered n - terminal region and a disordered c - terminal part . the former adopts a helix - turn - helix structure , in which the two -helices extend between residues 1418 and 2150 and are linked by a turn from residues 19 - 20 ; residues 437 are directly involved in binding the f1 domain . residues 418 , which are disordered in the dimer , are instead resolvable after binding . in the bound form , the inhibitory sequences of the two protomers in the dimer become closer ( their distance is shortened from 62   to 31   ) . this is possibly due to the flexibility of the c - terminal coiled coil region , which probably has a greater curvature in the complex . the long helix at the n - terminal region of if1 is inserted almost completely into the f1 domain ; only residues 4750 lie outside of it . this points to the central axis of the -subunit and forms a ~45 angle with it , heading from the external surface towards the central cavity . the contacts between the bovine inhibitor and the inhibited f1 domain are essentially located at the interface between the dp- and dp- subunits , even though if1 also contacts small portions of the  , e and tp- subunits . although the inhibitory sequence is comprised of residues 14 to 47 , this is not the only region that interacts with the f1fo - atpsynthase . in fact , as demonstrated by the time - dependent loss of inhibition seen by van raaij and co - workers , residues 113 and 4856 are important for stabilizing the structure ( the first peptide interacts directly with the f1 domain , while the second probably contributes to stabilize only the if1 dimer ) . with different approaches , found that a cluster of six residues , f22 , r25 , e26 , q27 , e30 and y33 in bovine if1 , is essential for the inhibitory activity of the peptide . in the crystal structure of the if1:f1 complex , the three core residues of the cluster , f22 , e26 and e30 , interact with three highly conserved residues of dp ( r408 , r412 , and e454 ) which are essential for the enzymatic catalysis and regulation of the f1fo - atpsynthase . e30 directly interacts with the -subunit of f1 and seems to be essential for the f1-if1 interaction ( figure 3 ) . interestingly , one of the three residues , e26 , is also implicated in the ph sensing mechanism of if1 . in 2008 , ando and ichikawa   discovered the key role of the glutamate residue in the ph - dependent activity of the inhibitory protein . they proposed that it was the high ph , by inducing the dissociation of the carboxyl group of e26 , to affect the conformation or direction of the side chain of the neighboring residue e30 , thus destabilizing the interaction between if1 and the f1 domain . as proposed by walker and co - workers , if1 seems to inhibit the hydrolysis of atp that occurs in mitochondria under hypoxic conditions by initially binding to the e/e - interface of the f1 domain , an event that appears to require the presence of atp in the active site . it was also suggested that the binding of atp induces a conformational change in the e - subunit , which creates the binding site for if1 . garcia and colleagues provided the first compelling evidences for a role of if1 in promoting the dimerization of the f1fo - atpsynthase , and for its involvement in the biogenesis of mitochondrial cristae . moreover , they also showed that , by increasing the expression of if1 in rat liver or as-30d hepatoma mitochondria , a rise in the dimer / monomer ratio of the f1fo - atpsynthase ( correlated with an increase in the enzyme activity ) is obtained , while the removal of the inhibitory protein from rat liver or bovine heart mitochondria resulted in a reduction of that ratio . the dimerization of the enzyme is essential for a correct biogenesis of mitochondrial cristae ; in fact , it represents a prerequisite for the generation of larger oligomers with a ribbon - like structure that promotes curvature and growth of tubular cristae membranes . in a recent study , we demonstrated the pivotal role of if1 in cell physiology through promotion of the f1fo - atpsynthase dimerization . briefly , we showed that if1 overexpression efficiently increase the activity and the ratio of dimeric to monomeric forms of the f1fo - atpsynthase , with augmented cristae number , mitochondrial membrane stability , and mitochondrial volume , thus ensuring a correct mitochondrial inner structure . decreased m induces the reversion of the f1fo - atpsynthase , which starts hydrolysing atp in the attempt to restore the h gradient through the mitochondrial inner membrane , transforming mitochondria in atp consumers ( figure 3(b ) ) . this condition associates with ischaemia , in which the interruption of tissue blood flow causes a reduction of cell oxygenation ( hypoxia ) inhibiting mitochondrial respiration . clear evidence for the reversal of the f1fo - atpsynthase during ischaemia can be obtained by using an etc inhibitor ( e.g. , rotenone , which acts on complex i , or nacn , which inhibits complex iv ) and concomitantly adding oligomycin , an antibiotic that blocks the f1fo - atpsynthase . these cells will experience a reduced depletion of atp compared to those bathed with the etc inhibitor but without oligomycin . the negative effect of the reversal of the f1fo - atpsynthase is coupled to the reversal of the adenosine nucleotide translocator ( ant ) , an imm transmembrane complex that , in physiological conditions , mediates the exchange of cytosolic adp and mitochondrial matrix atp ( utilizing the different gradients between the two compartments ) . reverse activities of both f1fo - atpsynthase and ant transform mitochondria from atp producers to atp consumers , leading to massive cytosolic atp depletion in hypoxic cells following ischaemia . reduced intracellular atp level is flanked by elevated cytoplasmic h , na , and ca concentrations , inducing osmotic loading and mitochondrial / endoplasmic reticulum injury so that , during ischaemia , death of hypoxic cells by necrosis easily occurs . early reperfusion minimizes the extent of cellular damage , salvaging cells within ischaemic regions from necrosis , but it can also causes lethal injury to cells with severe ischaemia - induced metabolic derangements ( reviewed in ) . in the latter case , reperfusion alters the activity of plasma membrane transporters ( e.g. , abolishing acidosis - mediated inhibition of the na - ca exchanger and inducing the activation of calpain , which disturbs na - k pump function ) , thus leading to massive influx of ca into the cytosol and mitochondrial ca overload . at the same time , resupply of oxygen to mitochondria restores atp production but also induces a rise in reactive oxygen species ( ros ) production . both mitochondrial ca overload and augmented ros levels represent the prelude to the opening of the mitochondrial permeability transition pore ( mptp ) and cell death . the entity of the reversal of the f1fo - atpsynthase and its action as an h motive atpase during oxygen deprivation were shown by jennings et al . in 1991 . studying the changes in atp depletion and anaerobic glycolysis in totally ischaemic dog heart after inhibiting the f1fo - atpsynthase via oligomycin , the same authors have evaluated that about 35% of atp utilization during the first 90 minutes of total ischaemia in the dog heart is due to the reversion of the enzyme activity . rouslin and colleagues   proved that the antibiotic has a very small and transient effect on mitochondrial function when used in fast heart - rate animals , like rats , if compared to slow heart - rate species , like larger mammals are . the same authors have subsequently shown that this diversity depends on the different f1fo - atpsynthase : if1 ratios , with a diverse ability to inhibit mitochondrial - driven consumption of atp when needed . the estimation was that if1 reduces the atp - hydrolysing activity of f1fo - atpsynthase during ischaemic conditions by up to 7080% ( in slow heart - rate species ) , thus preventing cellular damage due to ischaemic conditions and delaying cell death when oxygen and glucose are limited . upon reperfusion , the binding of if1 to the f1fo - atpsynthase is quickly reversed , so that sublethal ischaemic episodes could be followed by a relatively rapid recovery of intracellular atp . although this model is challenged by later evidence showing that in rat heart , during ischaemic preconditioning , mitochondrial atp hydrolysis is inhibited probably as a consequence of the binding of if1 , the variations in ratio between the enzyme and its controller among animal species are still a fascinating possibility . nonetheless , this ratio differs per se among organs and cell types of the same organ . by modulating the expression of if1 in human ( hela ) and murine ( c2c12 ) cells , we demonstrated that , when if1 is overexpressed , cells show a decrease in atp consumption . thus , variations in if1 expression could influence cellular or tissue resistance to ischaemic injury in different species or cell types . notably , if1 expression is elevated in highly oxidative cells , like neurons and kidney proximal tubules , which are highly susceptible to mitochondria deregulations . in the central nervous system , for example , neurons and astrocytes show a great difference in the if1  :  f1--subunit ratio , which is ~1.45 in the former and ~0.8 in the latter ; as a consequence , the inhibition of respiration with nacn causes a progressive loss of m in neurons , while in astrocytes the proton gradient is maintained at a new steady state . thus , higher levels of if1 could be advantageous in cells highly depending on oxidative phosphorylation by preventing atp depletion and quick cellular damage during ischaemia . a final interesting aspect is the highly probable involvement of if1 in the ischaemic preconditioning mechanism . this phenomenon , which is characterized by the acquirement of a strong resistance to ischaemia in tissue undergoing brief , repeated periods of sublethal ischaemia , is commonly observed in heart , skeletal muscle and brain . it is described as a slowing of energy metabolism with a decreased rate of atp depletion during ischaemia . if1 is proposed to take part in this process after the observation that rat heart preconditioning associates with the inhibition of mitochondrial atp hydrolysis during ischaemia . this was later confirmed by penna et al . , who showed that decrease in the enzyme 's activity after ischemic preconditioning correlates with an augmented binding of if1 . moreover , a consensus exists that the opening of mitochondrial atp - sensitive k channels plays a central role in cell protection during ischemic preconditioning , causing mitochondrial deenergisation and acidification owing to h / k exchange ; both effects are likely to promote the binding of if1 to the atp synthase . the importance of mitochondrial metabolism in cancer cells is underlined by the frequently observed , close interaction of glycolytic enzymes with mitochondria . this creates a mutually sustaining relationship between glycolysis , which represents the primary metabolic pathway for tumours sustenance , and oxidative phosphorylation . regarding the f1fo - atpsynthase endogenous regulator , if1 , its overexpression has been observed in many human carcinomas ( including lung , colon , breast , and cervix carcinomas , ehrlich ascites carcinoma , zajdela hepatoma and yoshida sarcoma ) , but little is still known about the associated effects , and the few theories that have been put forward are highly controversial . increased expression of the protein is associated with a higher binding efficiency to the f1fo - atpsynthase , suggesting a greater protection of cancer cells against energy dissipation upon f1fo - atpsynthase reversal . although more recent work has cast doubt on this hypothesis , revealing a relationship between the inhibitor overexpression in human carcinomas and an increase in both m and glycolytic rate . the protein may therefore be involved in protecting tumour cells from cytosolic atp depletion and excessive reactive oxygen species ( ros ) production ( the majority of tumours have little or no vascularization , so that cancerous cells grow in a hypoxic environment ) . over and above that , to guarantee cell viability , mitochondria should not become atp consumers . atp depletion , ros imbalance , low cytosolic ph and oxidation of nad(p)h facilitate the opening of the mitochondrial permeability transition pore ( mptp ) and the activation of the intrinsic apoptotic pathway . numerous studies have also demonstrated that transient hyperpolarization of the mitochondrial membrane can lead to cell apoptosis [ 38 , 95 ] . by preventing ros production and imm hyperpolarization if1 induces the dimerization of the f1fo - atpsynthase , which might play an essential role in preventing both mitochondrial network fragmentation and cytochrome c release from mitochondrial cristae , thus inhibiting the activation of the intrinsic apoptotic pathway . our previous studies seem to support these hypotheses , and , future and focused studies will shed light on this ( tan et al . . moreover , an original recent work by cuezva and co - workers has elegantly demonstrated that if1 is protective against chemotherapy and supports cell proliferation of cancerous cells via the nfkb pathway . although this work focuses principally on the postmitochondrial effects of if1 , it is anyway a compelling evidence for a contribution to neoplastic degeneration and resistance to apoptosis . a starting point to unravel how mitochondrial structure and function are primed by if1 overexpression , and to understand to what extent this dictates cellular transformation . to date , the absence of if1 has been correlated with only one human pathological condition of neuronal origin : a mitochondrial myopathy called luft 's disease , characterized by nonthyroidal hypermetabolism and densely packed mitochondrial cristae ( it is one of the rarest of the mitochondrial diseases , with only two reported cases ) . basal atpase activity in one of the two patients was seven times higher than normal , and no if1 activity was detected in fibroblasts cultured from the skeletal muscle ; however , no mutations in the atpif1 gene were identified , and the genetic cause of the disease remains obscure . despite what discussed above , we have very recently collected evidences for a deficiency in the atpif1 gene associated with a form of hypochromic anaemia ( shah et al . the mechanism we propose is related with the secondary effect that the absence of if1 , and the consequent lack in its inhibitory activity on the f1fo - atpsynthase reversion , has on m and matrix ph . it is known that erythroids ' differentiation is triggered by a decrease in m , that is responsible for a critical redistribution of intracellular ca and a transient activation of caspases . anyway , we found that the increase in mitochondrial matrix ph , which is observed in zebrafish models and murine cells carrying the mutated form of the atpif1 gene , is causally linked to a decrease in ferrochelatase activity , which leads to defects in the incorporation of fe into protoporphyrin ix to generate the hemoglobin prosthetic group heme . such a remarkable finding puts if1 amongst the regulators of heme biosynthesis , not only describing a new mechanism for sideroblastic anaemia , but also confirming the involvement of the inhibitory protein in human pathologies related to mitochondrial disorders . the f1fo - atpsynthase is a wonderful machinery , with the unique capacity of producing and consuming energy , if necessary , to preserve the integrity of the organelle to which it belongs . a precise and sustainable way to regulate its activity is therefore paramount , and the inhibitory factor 1 , a protein encoded by the nuclear dna , represents the molecule deputed to do so . in the face of a well - defined biochemistry , its role in cell physiology and mitochondrial anatomy has been only recently discovered , posing the protein at the cross - road between dynamics and energy balance . this , together with growing evidence for a contribution to cell and tissue pathology , leads to novel ways to investigate and thoroughly address if1 functional biology .
in mammals , the mitochondrial f1fo - atpsynthase sets out the energy homeostasis by producing the bulk of cellular atp . as for every enzyme , the laws of thermodynamics command it ; however , it is privileged to have a dedicated molecular regulator that controls its rotation . 
 this is the so - called atpase inhibitory factor 1 ( if1 ) that blocks its reversal to avoid the consumption of cellular atp when the enzyme acts as an atp hydrolase . 
 recent evidence has also demonstrated that if1 may control the alignment of the enzyme along the mitochondrial inner membrane , thus increasing the interest for the molecule . 
 we conceived this review to outline the fundamental knowledge of the f1fo - atpsynthase and link it to the molecular mechanisms by which if1 regulates its way of function , with the ultimate goal to highlight this as an important and possibly unique means to control this indispensable enzyme in both physiological and pathological settings .