Gene Conversions and Crossing over during Homologous and Homeologous Ectopic Recombination in Saccharomyces Cerevisiae

The pma1-105 mutation reduces the activity of the yeast plasma membrane H(+)-ATPase and causes cells to be both low pH and ammonium ion sensitive and resistant to the antibiotic hygromycin B. Revertants that can grow at pH 3.0 and on ammonium-containing plates frequently arise by ectopic recombination between pma1-105 and PMA2, a diverged gene that shares 85% DNA sequence identity with PMA1. The gene conversion tracts of revertants of pma1-105 were determined by DNA sequencing the hybrid PMA1::PMA2 genes. Gene conversion tracts ranged from 18-774 bp. The boundaries of these replacements were short (3-26 bp) regions of sequences that were identical between PMA1 and PMA2. These boundaries were not located at the regions of greatest shared identity between the two PMA genes. Similar results were obtained among low pH-resistant revertants of another mutation, pma1-147. One gene conversion was obtained in which the resulting PMA1::PMA2 hybrid was low pH-resistant but still hygromycin B-resistant. This partially active gene differs from a wild-type revertant only by the presence of two PMA2-encoded amino acid substitutions. Thus, some regions of PMA2 are not fully interchangeable with PMA1. We have also compared the efficiency of recombination between pma1-105 and either homeologous PMA2 sequence or homologous PMA1 donor sequences inserted at the same location. PMA2 X pma1-105 recombination occurred at a rate approximately 75-fold less than PMA1 X pma1-105 events. The difference in homology between the interacting sequences did not affect the proportion of gene conversion events associated with a cross-over, as in both cases approximately 5% of the Pma(+) recombinants had undergone reciprocal translocations between the two chromosomes carrying pma1-105 and the donor PMA sequences. Reciprocal translocations were identified by a simple and generally useful nutritional test.     

Evidence for coupling between membrane and cytoplasmic domains of the yeast plasma membrane H(+)-ATPase. An analysis of intragenic revertants of pma1-105.

A genetic approach was used to identify interacting portions of the plasma membrane H(+)-ATPase from Saccharomyces cerevisiae. The cellular sensitivity of the pma1-105 strain (S368F) to low external pH and to NH4+ was used to select intragenic revertants of two classes: phenotypically wild-type full revertants and partial revertants that were low pH-resistant but retained resistance to hygromycin B. All 10 full revertants had S368 restored. Among five partial revertants mapping to the original site within the phosphorylation domain, S368L and S368V were each found twice. One revertant contained an E367V substitution adjacent to the original S368F alteration. Four of 13 independently isolated second-site revertants mapped to one site, V289F, in the proposed phosphatase domain. Mutations within the proposed phosphatase and phosphorylation domains resulted in enzymes with increased vanadate sensitivity relative to the vanadate-insensitive S368F enzyme. These results suggest that sites S368, E367, and V289 contribute to a vanadate (Pi) binding domain or are able to interact with such a site within the catalytic domain. The remaining nine partial second-site revertants mapped to six sites within the putative transmembrane regions. Mutations within the transmembrane region had less of an effect on vanadate sensitivity. Most revertant enzymes showed small but significant increases in the rate of ATP hydrolysis relative to the S368F enzyme. Several enzymes no longer displayed the acid-sensitive pH-dependence seen in the S368F enzyme. These data provide novel evidence for an interaction between putative transmembrane helices 1-3 and 7 and the ATP hydrolytic portion of the enzyme.     

Membrane potential defect in hygromycin B-resistant pma1 mutants of Saccharomyces cerevisiae

The proton transport properties of hygromycin B-resistant pma1 mutants which show kinetic defects in the plasma membrane H+-ATPase were examined. It was found that net proton efflux, as measured by whole cell medium acidification in the presence of 25 mM KCl, was similar for normal and pma1 mutant cells. However, in the absence of added KCl, the extent of net proton efflux was considerably less in wild type than in pma1 mutant cells. The cellular membrane potential was implicated as an important factor in regulating net proton transport and was determined from [14C]tetraphenylphosphonium uptake studies to be considerably depolarized in the pma1 mutants. The growth of wild type cells, which is normally inhibited by hygromycin B at 200 micrograms/ml, was found to be resistant to the antibiotic by the addition of 50 mM KCl to the growth medium. These results suggest that the electrogenic behavior of proton transport by the H+-ATPase may be altered in pma1 mutants and that resistance to hygromycin B may be mediated via depolarization of the cellular membrane potential.     

Expression of the yeast plasma membrane [H+]ATPase in secretory vesicles. A new strategy for directed mutagenesis.

Secretory vesicles that accumulate in the temperature-sensitive sec6-4 strain of yeast have been shown to contain a vanadate-sensitive ATPase, presumably en route to the plasma membrane (Walworth, N. C., and Novick, P. J. (1987) J. Cell Biol. 105, 163-174). We have now established this enzyme to be a fully functional form of the PMA1 [H+]ATPase, identical in its catalytic properties to that found in the plasma membrane. In addition, the secretory vesicles are sealed tightly enough to permit the measurement of ATP-dependent proton pumping with fluorescent probes. We have gone on to exploit the vesicles as an expression system for site-directed mutants of the ATPase. For this purpose, a sec6-4 strain has been constructed in which the chromosomal PMA1 gene is under control of the GAL1 promoter; the mutant pma1 allele to be studied is introduced on a centromeric plasmid under the control of a novel heat shock promoter. In galactose medium at 23 degrees C, the wild-type ATPase is produced and supports normal vegetative growth. When the cells are switched to glucose medium at 37 degrees C, however, the wild-type gene turns off, the mutant gene turns on, and secretory vesicles accumulate. The vesicles contain a substantial amount of newly synthesized, plasmid-encoded ATPase (5-10% of total vesicle protein), but only traces of residual wild-type PMA1 ATPase and no detectable mitochondrial ATPase, vacuolar ATPase, or acid or alkaline phosphatase. To test the expression strategy, we have made use of pma1-105 (Ser368----Phe), a vanadate-resistant mutant previously characterized by standard methods (Perlin, D. S., Harris, S. L., Seto-Young, D., and Haber, J. E. (1989) J. Biol. Chem. 264, 21857-21864). In secretory vesicles, as expected, the plasmid-borne pma1-105 allele gives rise to a mutant enzyme with a reduced rate of ATP hydrolysis and a 100-fold increase in Ki for vanadate. Proton pumping is similarly resistant to vanadate. Thus, the vesicles appear well suited for the production and characterization of mutant forms of the PMA1 [H+]ATPase. They should also aid the study of other yeast membrane proteins that are essential for growth as well as heterologous proteins whose appearance in the plasma membrane may be toxic to the cell.     

The pma1 and pma2 H(+)-ATPases from Schizosaccharomyces pombe are functionally interchangeable.

The pma2 gene of Schizosaccharomyces pombe codes for a polypeptide having a predicted Mr of 110,126 and which is 79% identical to the plasma membrane H(+)-ATPase encoded by the pma1 gene. The pma2 gene, unlike pma1, is weakly expressed and not essential to mitotic growth. By constructing yeast strains in which the chromosomal pma2 gene is under control of the adh promoter, it has been possible to identify the overproduced ATPase in plasma membrane via formation of a phosphoenzyme. In a pma1-1 mutant strain whose ATPase activity is insensitive to vanadate, the overexpressed pma2 gene restores vanadate sensitivity. It also rescues a pma1 null mutant from lethality. These results demonstrate that the two H(+)-ATPases are functionally interchangeable in vivo but differently expressed.     

Pleiotropic plasma membrane ATPase mutations of Saccharomyces cerevisiae.

We isolated a large number of mutations in the structural gene for the plasma membrane ATPase (PMA1) of Saccharomyces cerevisiae. These mutations were selected by their resistance to the aminoglycoside antibiotic hygromycin B. Biochemical analysis of purified membrane preparations showed that the plasma membrane ATPase activity of the mutants was reduced as much as 75%. Intragenic complementation of pma1 mutants suggested that the yeast plasma membrane ATPase was a multimeric enzyme. The pma1 mutants were apparently defective in maintaining internal pH; more than half of the mutants were unable to grow either at a low pH or in the presence of a weak acid. Most pma1 mutants were also osmotic pressure sensitive. At a very low temperature (5 degrees C) many pma1 mutants were unable to grow and were arrested as unbudded cells. The three most severely affected mutants were also unable to grow in the presence of NH4+. The most extreme mutant exhibited a severe defect in progression through the cell cycle; on synthetic medium, the cells progressively accumulated nucleus-containing small buds that generally failed to complete bud enlargement and cytokinesis. Most of the pleiotropic phenotypes of pma1 mutants could be suppressed by the addition of 50 mM KCl but not NaCl to the medium.     

After chitin docking, toxicity of Kluyveromyces lactis zymocin requires Saccharomyces cerevisiae plasma membrane H+-ATPase

Zymocin, a three-subunit (alpha beta gamma) toxin complex from Kluyveromyces lactis, imposes a cell cycle block on Saccharomyces cerevisiae. Phenotypic analysis of the resistant kti10 mutant implies a membrane defect, suggesting that KTI10 represents a gene involved early in the zymocin response. Consistently, KTI10 is shown here to be allelic to PMA1 encoding H(+)-ATPase, a plasma membrane H(+) pump vital for membrane energization (Delta Psi). Like pma1 mutants, kti10 cells lose viability at low pH, indicating a pH homeostasis defect, and resist the antibiotic hygromycin B, uptake of which is known to be Pma1 and Delta Psi sensitive. Similar to kti10 cells, pma1 mutants with reported H(+) pump defects survive in the presence of exozymocin but do not resist endogenous expression of its lethal gamma-toxin subunit. Based on DNA sequence data, kti10 cells are predicted to produce a malfunctional Pma1 variant with expression levels that are normal. Intriguingly, zymocin protection of kti10 cells is suppressed by excess H(+), a scenario ineffective in bypassing resistance of chitin or toxin target mutants. Together with unaltered zymocin docking and gamma-toxin import events in kti10 cells, our data suggest that Pma1's role in zymocin action is likely to involve activation of gamma-toxin in a step following its cellular uptake.     

A single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma-32P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors (Ulaszewski, S., Coddington, A., and Goffeau, A. (1986) Curr. Genet. 10, 359-364).     

Altered plasma membrane H(+)-ATPase from the Dio-9-resistant pma1-2 mutant of Schizosaccharomyces pombe.

The pma1-2 mutation affecting the plasma membrane H(+)-ATPase of Schizosaccharomyces pombe has been selected for resistance to the antibiotic Dio-9. In membrane fractions purified from glucose-starved cells, the mutant ATPase activity is reduced by 96%, is insensitive to inhibition by vanadate and has a pH profile displaced in the acidic pH range when compared to the wild type. The maximum velocity of the H(+)-ATPase activity of plasma membranes from glucose-activated pma1-2 cells is activated 20-fold. This is in striking contrast with the wild-type ATPase activity, the maximal velocity of which is not affected by glucose. However, similar to the wild-type enzyme, glucose activation of the pma1-2 mutant H(+)-ATPase reduces the Km for MgATP 9-2 mM and shifts the optimal pH from 4.8 to 6.0-6.5. The pma1-2 mutation modifies Lys250 to a threonine, which is highly conserved in fungal and plant H(+)-ATPases. These results, compared to those reported for mutations of neighbour residues in yeast or mammalian P-type ATPases, suggest that Lys250 could play a significant role, not only in phosphate binding and/or in the E1P-E2P conformational isomerisation, but also in glucose activation of the H(+)-ATPase.     

Effects of phosphate and hydrophobic molecules on two mutations in the beta-strand sector of the H(+)-ATPase from the yeast plasma membrane

At concentrations from 10 to 100 mM, inorganic phosphate and sulfate stimulate the activity of the H(+)-ATPase purified from the wild type Schizosaccharomyces pombe plasma membranes. Compared to the wild type ATPase, the stimulation by phosphate is more pronounced in the mutant pma1-1 (Gly-268----Asp) and is much reduced in the mutant pma1-2 (Lys-250----Thr) enzymes. In contrast, the inhibition by trifluoperazine is less pronounced in the pma1-1 mutant than in the wild type or pma1-2 mutant. The mutant pma1-2 ATPase activity is markedly stimulated by 10-20% dimethyl sulfoxide, which has a limited effect on the wild type and pma1-1 enzymes. These data indicate that the protein domain located in the beta-strand sector, including Lys-250 and Gly-268, is located in the active site and that its hydrophobic character influences the interactions of the yeast H(+)-ATPase with inorganic phosphate, as well as with the hydrophobic inhibitor trifluoperazine or the hydrophobic solvent dimethyl sulfoxide.     

Molecular cloning of a family of plant genes encoding a protein homologous to plasma membrane H+-translocating ATPases

cDNA clones from Nicotiana plumbaginifolia have been isolated by hybridization to a yeast H+-ATPase gene. The largest one encodes a polypeptide (PMA2) of 956 amino acid residues which exhibits a homology of 73% with a limited protein sequence obtained from purified oat plasma membrane H+-ATPase (Schaller and Sussman, Plant Physiol. 86, 512-516, 1988) and an 82% homology with the Arabidopsis thaliana pma gene (Harper et al., Proc. Natl. Acad. Sci. USA 86, 1234-1238). It is therefore concluded that the N. plumbaginifolia pma2 gene encodes a plasma membrane H+-ATPase. Southern blot hybridization indicates that the plant pma2 gene belongs to a multigene family. Partial sequences of cDNA clones show that at least three pma genes are expressed in root cells.     

Characterization of an allele-nonspecific intragenic suppressor in the yeast plasma membrane H+-ATPase gene (Pma1).

We have analyzed the ability of A165V, V169I/D170N, and P536L mutations to suppress pma1 dominant lethal alleles and found that the P536L mutation is able to suppress the dominant lethality of the pma1-R271T, -D378N, -D378E, and -K474R mutant alleles. Genetic and biochemical analyses of site-directed mutants at Pro-536 suggest that this amino acid may not be essential for function but is important for biogenesis of the ATPase. Proteins encoded by dominant lethal pma1 alleles are retained in the endoplasmic reticulum, thus interfering with transport of wild-type Pma1. Immunofluorescence studies of yeast conditionally expressing revertant alleles show that the mutant enzymes are correctly located at the plasma membrane and do not disturb targeting of the wild-type enzyme. We propose that changes in Pro-536 may influence the folding of the protein encoded by a dominant negative allele so that it is no longer recognized and retained as a misfolded protein by the endoplasmic reticulum.     

Effects of mutations of conserved Lys-155 and Thr-156 residues in the phosphate-binding glycine-rich sequence of the F1-ATPase beta subunit of Escherichia coli.

beta Lys-155 in the glycine-rich sequence of the beta subunit of Escherichia coli F1-ATPase has been shown to be near the gamma-phosphate moiety of ATP by affinity labeling (Ida, K., Noumi, T., Maeda, M., Fukui, T., and Futai, M. (1991) J. Biol. Chem. 266, 5424-5429). For examination of the roles of beta Lys-155 and beta Thr-156, mutants (beta Lys-155-->Ala, Ser, or Thr; beta Thr-156-->Ala, Cys, Asp, or Ser; beta Lys-155/beta Thr-156-->beta Thr-155/beta Lys-156; and beta Thr-156/beta Val-157-->beta Ala-156/beta Thr-157) were constructed, and their properties were studied extensively. The beta Ser-156 mutant was active in ATP synthesis and had approximately 1.5-fold higher membrane ATPase activity than the wild type. Other mutants were defective in ATP synthesis, had < 0.1% of the membrane ATPase activity of the wild type, and showed no ATP-dependent formation of an electrochemical proton gradient. The mutants had essentially the same amounts of F1 in their membranes as the wild type. Purified mutant enzymes (beta Ala-155, beta Ser-155, beta Ala-156, and beta Cys-156) showed low rates of multisite (< 0.02% of the wild type) and unisite (< 1.5% of the wild type) catalyses. The k1 values of the mutant enzymes for unisite catalysis were lower than that of the wild type: not detectable with the beta Ala-156 and beta Cys-156 enzymes and 10(2)-fold lower with the beta Ala-155 and beta Ser-155 enzymes. The beta Thr-156-->Ala or Cys enzyme showed an altered response to Mg2+, suggesting that beta Thr-156 may be closely related to Mg2+ binding. These results suggest that beta Lys-155 and beta Thr-156 are essential for catalysis and are possibly located in the catalytic site, although beta Thr-156 could be replaced by a serine residue.     

Efficient degradation of misfolded mutant Pma1 by endoplasmic reticulum-associated degradation requires Atg19 and the Cvt/autophagy pathway

Misfolded proteins are usually arrested in the endoplasmic reticulum (ER) and degraded by the ER-associated degradation (ERAD) machinery. Several mutant alleles of PMA1, the gene coding for the plasma membrane H(+)-ATPase, render misfolded proteins that are retained in the ER and degraded by ERAD. A subset of misfolded PMA1 mutants exhibit a dominant negative effect on yeast growth since, when coexpressed with the wild-type allele, both proteins are retained in the ER. We have used a pma1-D378T dominant negative mutant to identify new genes involved in ERAD. A genetic screen was performed for isolation of multicopy suppressors of a GAL1-pma1-D378T allele. ATG19, a member of the cytoplasm to vacuole targeting (Cvt) pathway, was found to suppress the growth arrest phenotype caused by the expression of pma1-D378T. ATG19 accelerates the degradation of pma1-D378T thus allowing the co-retained wild-type Pma1 to reach the plasma membrane. ATG19 was also able to suppress other dominant lethal PMA1 mutations. The degradation of the mutant ATPase occurs in the proteasome and requires intact both ERAD and Cvt/autophagy pathways. We propose the cooperation of both pathways for an efficient degradation of misfolded Pma1.     

Post-translational modification of plant plasma membrane H(+)-ATPase as a requirement for functional complementation of a yeast transport mutant.

Many heterologous membrane proteins expressed in the yeast Saccharomyces cerevisiae fail to reach their normal cellular location and instead accumulate in stacked internal membranes. Arabidopsis thaliana plasma membrane H(+)-ATPase isoform 2 (AHA2) is expressed predominantly in yeast internal membranes and fails to complement a yeast strain devoid of its endogenous H(+)-ATPase Pma1. We observed that phosphorylation of AHA2 in the heterologous host and subsequent binding of 14-3-3 protein is crucial for the ability of AHA2 to substitute for Pma1. Thus, mutants of AHA2, complementing pma1, showed increased phosphorylation at the penultimate residue (Thr(947)), which creates a binding site for endogenous 14-3-3 protein. Only a pool of ATPase in the plasma membrane is phosphorylated. Double mutants carrying in addition a T947A substitution lost their ability to complement pma1. However, mutants affected in both autoinhibitory regions of the C-terminal regulatory domain complemented pma1 irrespective of their ability to become phosphorylated at Thr(947). This demonstrates that it is the activity status of the mutant enzyme and neither redirection of trafficking nor 14-3-3 binding per se that determines the ability of H(+)-pumps to rescue pma1.     

A second transport ATPase gene in Saccharomyces cerevisiae

A second transport ATPase gene from Saccharomyces cerevisiae has been identified by hybridization to a PMA1 probe and sequenced. The gene called PMA2 encodes a polypeptide of Mr = 102,157, which, with the exception of the 144 amino-terminal residues, is highly homologous to the structural gene PMA1 for the H+-ATPase. It is localized on the chromosome XVI at 16.7 centimorgan from gal4 and is not essential for haploid growth. Comparison between the upstream, noncoding DNA regions of PMA1 and PMA2 indicates that the two genes are controlled differently. The extensive amino acid sequence homology with the fungal H+-ATPases described so far indicates that the PMA2-encoded protein is also able to function as a H+ pump. This is supported by the observation that in pma1 mutants with reduced plasma membrane ATPase activity, disruption of the PMA2 gene confers the ability to grow under alkaline pH conditions. Slower development of diploids is also observed on normal minimal medium after bilateral disruption of PMA2 in the two parents.     

Activity of the plasma membrane H(+)-ATPase and optimal glycolytic flux are required for rapid adaptation and growth of Saccharomyces cerevisiae in the presence of the weak-acid preservative sorbic acid.

The weak acid sorbic acid transiently inhibited the growth of Saccharomyces cerevisiae in media at low pH. During a lag period, the length of which depended on the severity of this weak-acid stress, yeast cells appeared to adapt to this stress, eventually recovering and growing normally. This adaptation to weak-acid stress was not due to metabolism and removal of the sorbic acid. A pma1-205 mutant, with about half the normal membrane H+-ATPase activity, was shown to be more sensitive to sorbic acid than its parent. Sorbic acid appeared to stimulate plasma membrane H+-ATPase activity in both PMA1 and pma1-205. Consistent with this, cellular ATP levels showed drastic reductions, the extent of which depended on the severity of weak-acid stress. The weak acid did not appear to affect the synthesis of ATP because CO2 production and O2 consumption were not affected significantly in PMA1 and pma1-205 cells. However, a glycolytic mutant, with about one-third the normal pyruvate kinase and phosphofructokinase activity and hence a reduced capacity to generate ATP, was more sensitive to sorbic acid than its isogenic parent. These data are consistent with the idea that adaptation by yeast cells to sorbic acid is dependent on (i) the restoration of internal pH via the export of protons by the membrane H+-ATPase in an energy-demanding process and (ii) the generation of sufficient ATP to drive this process and still allow growth.     

Ouabain-insensitive Na-ATPase activity in the basolateral membrane from rat jejunum

1. In the basolateral membrane preparation of the rat enterocyte (jejunal tract) there is not only the well-known (Na,K)-ATPase activity, but also a ouabain-insensitive Na-ATPase. 2. The Na-ATPase is not activated by anions or other monovalent cations. As a substrate, ATP cannot be replaced by other nucleotides. 3. The Na-ATPase is insensitive to ouabain and bumetanide, inhibited partially by furosemide and totally by ethacrynate. 4. The activation of Na-ATPase at different Na concentrations shows an hyperbolic curve (Km = 15.7 +/- 2.3 mM and Vmax = 204 +/- 19 nmoles Pi/mg protein per min) different from the sigmoidal curve (Km = 9.8 +/- 1.2 mM and Vmax = 640 +/- 15 nmoles Pi/mg protein per min) shown by (Na,K)-ATPase. 5. These results are compared with the corresponding ones found in other animals and tissues in which the Na-ATPase was found. 6. The Na-ATPase activity can be interpreted as the enzymatic correspondent of a ouabain-insensitive Na pump, present in the basolateral membrane of the enterocyte different in behaviour with respect to the known Na pump.     

The transmembrane prolines of the mitochondrial ADP/ATP carrier are involved in nucleotide binding and transport and its biogenesis

The mitochondrial ADP/ATP carrier (Ancp) is a paradigm of the mitochondrial carrier family, which allows cross-talk between mitochondria, where cell energy is mainly produced, and cytosol, where cell energy is mainly consumed. The members of this family share numerous structural and functional characteristics. Resolution of the atomic structure of the bovine Ancp, in a complex with one of its specific inhibitors, revealed interesting features and suggested the involvement of some particular residues in the movements of the protein to perform translocation of nucleotides from one side of the membrane to the other. They correspond to three prolines located in the odd-numbered transmembrane helices (TMH), Pro-27, Pro-132, and Pro-229. The corresponding residues of the yeast Ancp (Pro-43, Ser-147, and Pro-247) were mutated into alanine or leucine, one at a time and analysis of the various mutants evidenced a crucial role of Pro-43 and Pro-247 during nucleotide transport. Beside, replacement of Ser-147 with proline does not inactivate Ancp and this is discussed in view of the conservation of the three prolines at equivalent positions in the Ancp sequences. These prolines belong to the signature sequences of the mitochondrial carriers and we propose they play a dual role in the mitochondrial ADP/ATP carrier function and biogenesis. Unexpectedly their mutations cause more general effects on mitochondrial biogenesis and morphology, as evidenced by measurements of respiratory rates, cytochrome contents, and also clearly highlighted by fluorescence microscopy.     

Ca2+-dependent ATPases in the basolateral membrane of rat kidney cortex

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.