Ion permeability changes involving ion channels and transporters are essential for fertilization,... more Ion permeability changes involving ion channels and transporters are essential for fertilization, since they are key elements in sperm-egg signaling and environmental sensing. Changes modulated by external factors and components from the outer layer of the homologous egg, like the sperm-activating peptides and the fucose-sulfate glycoconjugate, trigger complex signaling systems in the sperm that modulate how sperm swim, find the egg and fertilize it. These signa-ling systems depend on plasma membrane ion permeability and involve an alteration of second messenger levels, changes of membrane potential and intracellular Ca 2+ , Na + and pH, along with changes in sperm morphology in the case of the acrosome reaction (AR). New procedures to elucidate the signaling pathways implicated in sperm ion transport have expanded the opportunities to dissect sperm-egg signaling revealing how sperm ion channels participate in activation, motility, chemotaxis, and the AR. The sperm signaling pathways involve a large variety of ion channels and transporters, which are discretely localized and finely orchestrated to play specific roles that define the elaborate performance of sperm and allow a successful fertilization. In this review we will focus on the signaling molecules involved in the final aim of the sperm, to reach and fertilize the egg.
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm– egg signaling and unveil novel contraception targets.
Society of Reproduction and Fertility supplement, 2007
Spermatozoa depend upon ion channels to rapidly exchange information with the outside world and t... more Spermatozoa depend upon ion channels to rapidly exchange information with the outside world and to fertilise the egg. These efficient ion transporters participate in many of the most important sperm processes, such as motility and capacitation. It is well known that sperm swimming is regulated by [Ca2+]i. In the sea urchin sperm speract, a decapeptide isolated from egg outer envelope, induces changes in intracellular Ca2+ ([Ca2+]i), Na+, cAMP and cGMP, membrane potential (Em) and pH (pHi). Photoactivation of a speract analogue induces Ca2+ fluctuations that generate turns that are followed by straighter swimming paths. A fast component of the [Ca2+], increase that most likely occurs through voltage dependent Ca2+ channels (Ca(v)s) is essential for these turns. The Ca(v)s involved are modulated by the Em changes triggered by speract. On the other hand, mammalian sperm gain the ability to fertilise the egg after undergoing a series of physiological changes in the female tract. This ma...
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets. Reproduction (2006) 131 977-988 q 2006 Society for Reproduction and Fertility
Proton H+-ATPases are among the most fundamental enzymes in nature. They are present in every kno... more Proton H+-ATPases are among the most fundamental enzymes in nature. They are present in every known living cell, and they function in biological energy transduction and pH homeostasis. ATP-dependent proton pumping is performed by two distinct mechanisms. ...
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2014
Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentra... more Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are among the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO 3 − it is able to translate CO 2 /HCO 3 − /pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130 years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
Biochemical and Biophysical Research Communications, 2004
The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR req... more The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome
Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many ... more Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn(2+) was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn(2+) change membrane potential (Em) and increase the concentration of intracellular Ca(2+) ([Ca(2+)]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn(2+) response in sperm of this species mainly involves an Em hyperpolarization caused by K(+) channel activation. The pharmacological profile of the Zn(2+)-induced hyperpolarization indicates that the cGMP-gated K(+) selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn(2+). Considering that Zn(2+) also induces [Ca(2+)]i fluctuations, our observations suggest that Zn(2+) activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn(2+) in male gamete function.
Background: The transient receptor potential channel (TRP) family includes more than 30 proteins;... more Background: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca 2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca 2+ ([Ca 2+ ]i). Ca 2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored.
Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertio... more Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+ -selective channel was confirmed. Here, it is reported that the Ca2+ -selective channel from mouse sperm plasma membrane displayed a PNa+/PK+ = 1.6 +/- 0.2 (n = 4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/PK+ = 2.5 +/- 0.3, n = 3) with a main open conductance substate of 103 pS and a smaller open substate of 51 pS (600 mM K+ cis/100 mM Na+ trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 +/- 0.02, n = 3), and a low activity mode (open probability < 1%, n = 4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 pS (600 KCl cis/100 KCl trans), was identified. It displayed a high, nearly constant open probability (approximately 0.90) in the 0 to -80 mV range.
• Ion channels participate in crucial sperm functions such as motility, capacitation and the acro... more • Ion channels participate in crucial sperm functions such as motility, capacitation and the acrosome reaction. • Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology. • We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa to investigate their ion channels. • This work presents the first evidence for the presence of calcium-dependent chloride channels (CaCCs) in human spermatozoa; they could be constituted by TMEM16. • The CaCCs play an important role in the physiology of human spermatozoa and participate in the acrosome reaction.
The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria ... more The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4pmol min-' mg-' to 6-7pmol min-' mg-' upon incubation for 5 -6 h at 38 'C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762-37691. At maximal activation approximately 50 % of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 12'I-labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, 1 H* and IS* of the process were 53.34 kJ/mol, 50.83 kJ/mol and -158.99 J mol-K -l , respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets. Reproduction (2006) 131 977-988 q 2006 Society for Reproduction and Fertility
Proceedings of the National Academy of Sciences, 1991
Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were as... more Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-["4Cdicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of-the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.
Ion permeability changes involving ion channels and transporters are essential for fertilization,... more Ion permeability changes involving ion channels and transporters are essential for fertilization, since they are key elements in sperm-egg signaling and environmental sensing. Changes modulated by external factors and components from the outer layer of the homologous egg, like the sperm-activating peptides and the fucose-sulfate glycoconjugate, trigger complex signaling systems in the sperm that modulate how sperm swim, find the egg and fertilize it. These signa-ling systems depend on plasma membrane ion permeability and involve an alteration of second messenger levels, changes of membrane potential and intracellular Ca 2+ , Na + and pH, along with changes in sperm morphology in the case of the acrosome reaction (AR). New procedures to elucidate the signaling pathways implicated in sperm ion transport have expanded the opportunities to dissect sperm-egg signaling revealing how sperm ion channels participate in activation, motility, chemotaxis, and the AR. The sperm signaling pathways involve a large variety of ion channels and transporters, which are discretely localized and finely orchestrated to play specific roles that define the elaborate performance of sperm and allow a successful fertilization. In this review we will focus on the signaling molecules involved in the final aim of the sperm, to reach and fertilize the egg.
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm– egg signaling and unveil novel contraception targets.
Society of Reproduction and Fertility supplement, 2007
Spermatozoa depend upon ion channels to rapidly exchange information with the outside world and t... more Spermatozoa depend upon ion channels to rapidly exchange information with the outside world and to fertilise the egg. These efficient ion transporters participate in many of the most important sperm processes, such as motility and capacitation. It is well known that sperm swimming is regulated by [Ca2+]i. In the sea urchin sperm speract, a decapeptide isolated from egg outer envelope, induces changes in intracellular Ca2+ ([Ca2+]i), Na+, cAMP and cGMP, membrane potential (Em) and pH (pHi). Photoactivation of a speract analogue induces Ca2+ fluctuations that generate turns that are followed by straighter swimming paths. A fast component of the [Ca2+], increase that most likely occurs through voltage dependent Ca2+ channels (Ca(v)s) is essential for these turns. The Ca(v)s involved are modulated by the Em changes triggered by speract. On the other hand, mammalian sperm gain the ability to fertilise the egg after undergoing a series of physiological changes in the female tract. This ma...
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets. Reproduction (2006) 131 977-988 q 2006 Society for Reproduction and Fertility
Proton H+-ATPases are among the most fundamental enzymes in nature. They are present in every kno... more Proton H+-ATPases are among the most fundamental enzymes in nature. They are present in every known living cell, and they function in biological energy transduction and pH homeostasis. ATP-dependent proton pumping is performed by two distinct mechanisms. ...
Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2014
Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentra... more Fertilization, a key step in sexual reproduction, requires orchestrated changes in cAMP concentrations. It is notable that spermatozoa (sperm) are among the cell types with extremely high adenylyl cyclase (AC) activity. As production and consumption of this second messenger need to be locally regulated, the discovery of soluble AC (sAC) has broadened our understanding of how such cells deal with these requirements. In addition, because sAC is directly regulated by HCO 3 − it is able to translate CO 2 /HCO 3 − /pH changes into cAMP levels. Fundamental sperm functions such as maturation, motility regulation and the acrosome reaction are influenced by cAMP; this is especially true for sperm of the sea urchin (SU), an organism that has been a model in the study of fertilization for more than 130 years. Here we summarize the discovery and properties of SU sperm sAC, and discuss its involvement in sperm physiology. This article is part of a Special Issue entitled: The role of soluble adenylyl cyclase in health and disease.
Biochemical and Biophysical Research Communications, 2004
The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR req... more The sperm acrosome reaction (AR) involves ion channel activation. In sea urchin sperm, the AR requires Ca2+ and Na+ influx and K+ and H+ efflux. During the AR, the plasma membrane fuses with the acrosomal vesicle membrane forming hybrid membrane vesicles that are released from sperm into the medium. This paper reports the isolation and preliminary characterization of these acrosome
Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many ... more Zinc (Zn(2+)) has been recently recognized as a crucial element for male gamete function in many species although its detailed mechanism of action is poorly understood. In sea urchin spermatozoa, Zn(2+) was reported as an essential trace ion for efficient sperm motility initiation and the acrosome reaction by modulating intracellular pH (pHi). In this study we found that submicromolar concentrations of free Zn(2+) change membrane potential (Em) and increase the concentration of intracellular Ca(2+) ([Ca(2+)]i) and cAMP in Lytechinus pictus sperm. Our results indicate that the Zn(2+) response in sperm of this species mainly involves an Em hyperpolarization caused by K(+) channel activation. The pharmacological profile of the Zn(2+)-induced hyperpolarization indicates that the cGMP-gated K(+) selective channel (tetraKCNG/CNGK), which is crucial for speract signaling, is likely a main target for Zn(2+). Considering that Zn(2+) also induces [Ca(2+)]i fluctuations, our observations suggest that Zn(2+) activates the signaling cascade of speract, except for an increase in cGMP, and facilitates sperm motility initiation upon spawning. These findings provide new insights about the role of Zn(2+) in male gamete function.
Background: The transient receptor potential channel (TRP) family includes more than 30 proteins;... more Background: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca 2+ dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca 2+ ([Ca 2+ ]i). Ca 2+ triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored.
Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertio... more Fusion of purified mouse sperm plasma membranes to planar lipid bilayers resulted in the insertion of three ion channel types. They could be discerned on the basis of their selectivity, conductance, gating and voltage-dependent properties. The presence of a previously reported large, Ca2+ -selective channel was confirmed. Here, it is reported that the Ca2+ -selective channel from mouse sperm plasma membrane displayed a PNa+/PK+ = 1.6 +/- 0.2 (n = 4) and was blocked by micromolar concentrations of ruthenium red. Fusion yielded also a cation-selective channel (PNa+/PK+ = 2.5 +/- 0.3, n = 3) with a main open conductance substate of 103 pS and a smaller open substate of 51 pS (600 mM K+ cis/100 mM Na+ trans). The channel inserted into bilayers in two stable fashions: a high-activity mode (open probability = 0.57 +/- 0.02, n = 3), and a low activity mode (open probability < 1%, n = 4). In high mode, the channel displayed bursting kinetics and burst length was voltage independent. In addition, a perfectly anion-selective channel, with a slope conductance of 83 pS (600 KCl cis/100 KCl trans), was identified. It displayed a high, nearly constant open probability (approximately 0.90) in the 0 to -80 mV range.
• Ion channels participate in crucial sperm functions such as motility, capacitation and the acro... more • Ion channels participate in crucial sperm functions such as motility, capacitation and the acrosome reaction. • Chloride, the main anion in physiological solutions, is deeply involved in sperm physiology. • We implemented a modified perforated patch-clamp strategy to obtain whole cell recordings sealing on the head of mature human spermatozoa to investigate their ion channels. • This work presents the first evidence for the presence of calcium-dependent chloride channels (CaCCs) in human spermatozoa; they could be constituted by TMEM16. • The CaCCs play an important role in the physiology of human spermatozoa and participate in the acrosome reaction.
The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria ... more The rate of ATP hydrolysis by submitochondrial particles prepared from bovine heart mitochondria in the presence of Mg2+ and ATP increases from a value of 0.4pmol min-' mg-' to 6-7pmol min-' mg-' upon incubation for 5 -6 h at 38 'C. The increase in activity does not occur in particles that have been passed through a Sephadex column. The activation is prevented and partially reversed by ATP. This indicates that the increase in hydrolytic activity is due to abolition of the inhibitory action of the natural ATPase inhibitor protein of Pullman and Monroy [(1963) J. Biol. Chem. 238, 3762-37691. At maximal activation approximately 50 % of the inhibitor protein of the starting preparation remains in the particles as inferred from direct assay of inhibitor protein content and by its interaction with 12'I-labeled antibodies directed against the inhibitor protein. The extent of the activation, which presumably is an index of the equilibrium between active and inactive enzymes, is strictly dependent on salts. The rate of the activation depends on the concentration of salts and is favored by alkaline pH. From results of experiments on the effect of temperature on the rate of activation of the ATPase, it was calculated that the activation energy, 1 H* and IS* of the process were 53.34 kJ/mol, 50.83 kJ/mol and -158.99 J mol-K -l , respectively. The data indicate that in its native inhibiting state, the interaction of the inhibitor protein with the enzyme involves electrostatic interactions. Also it is concluded that abolition of the inhibitory action of the protein on ATPase activity is not compulsorily linked to release of the protein into the water space.
Ion channels are extraordinarily efficient machines that move ions in diversely controlled manner... more Ion channels are extraordinarily efficient machines that move ions in diversely controlled manners, allowing cells to rapidly exchange information with the outside world and with other cells. Communication is the currency of fertilization, as it is of most fundamental cell signaling events. Ion channels are deeply involved in the dialogue between sperm, its surroundings, and the egg. How sperm swim, find the egg and fertilize it depend on ion permeability changes modulated by environmental cues and components of the egg outer layer. Different ion channels distinctly localized in these tiny, amazing cells perform specific decoding functions that shape the sophisticated behavior of sperm. It is not surprising that certain sperm ion channels are turning out to be unique. New strategies to characterize sperm ion transport have opened exciting possibilities to dissect sperm-egg signaling and unveil novel contraception targets. Reproduction (2006) 131 977-988 q 2006 Society for Reproduction and Fertility
Proceedings of the National Academy of Sciences, 1991
Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were as... more Yeast mutants in which genes encoding subunits of the vacuolar H+-ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N'-["4Cdicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. We conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of-the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.
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Papers by Carmen Beltrán