Knockdown of DAPIT (diabetes-associated protein in insulin-sensitive tissue) results in loss of ATP synthase in mitochondria

It was found recently that a diabetes-associated protein in insulin-sensitive tissue (DAPIT) is associated with mitochondrial ATP synthase. Here, we report that the suppressed expression of DAPIT in DAPIT-knockdown HeLa cells causes loss of the population of ATP synthase in mitochondria. Consequently, DAPIT-knockdown cells show smaller mitochondrial ATP synthesis activity, slower growth in normal medium, and poorer viability in glucose-free medium than the control cells. The mRNA levels of α- and β-subunits of ATP synthase remain unchanged by DAPIT knockdown. These results indicate a critical role of DAPIT in maintaining the ATP synthase population in mitochondria and raise an intriguing possibility of active role of DAPIT in cellular energy metabolism.     

Identification of two proteins associated with mammalian ATP synthase

Bovine mitochondrial ATP synthase commonly is isolated as a monomeric complex that contains 16 protein subunits and the natural IF(1) inhibitor protein in substoichiometric amounts. Alternatively ATP synthase can be isolated in dimeric and higher oligomeric states using digitonin for membrane solubilization and blue native or clear native electrophoresis for separation of the native mitochondrial complexes. Using blue native electrophoresis we could identify two ATP synthase-associated membrane proteins with masses smaller than 7 kDa and isoelectric points close to 10 that previously had been removed during purification. We show that in the mitochondrial membrane both proteins are almost quantitatively bound to ATP synthase. Both proteins had been identified earlier in a different context, but their association with ATP synthase was unknown. The first one had been named 6.8-kDa mitochondrial proteolipid because it can be isolated by chloroform/methanol extraction from mitochondrial membranes. The second one had been denoted as diabetes-associated protein in insulin-sensitive tissue (DAPIT), which may provide a clue for further functional and clinical investigations.     

Regulation of phospholipid-ATPase complex interaction by the adenine nucleotide carrier

(1) The effect of phospholipids on a preparation containing the ATPase complex and the adenine nucleotide carrier is studied in the presence of ligands known to affect the conformation of these components of the mitochondrial inner membrane. (2) When ATPase activity is abolished by phospholipid depletion, the reactivation induced by phosphatidylcholine is prevented by the simultaneous addition of ATP. ADP partially reproduces the ATP effect. AMP, GTP, UTP and P_i are ineffective. (3) The influence of ATP is associated with reduced phospholipid binding to the membrane fragments and is reversible. The ATP effect on reconstitution is not manifest when phosphatidylcholine is added together with negatively charged phospholipids. (4) Carboxyatractyloside does not modify the phospholipid-ATPase complex interaction but bongkrekic acid is as effective as ATP. In the presence of ADP, the influence of bongkrekic acid is considerably increased. (5) It is concluded that the binding of ATP to the adenine nucleotide carrier enables the complex to select between the charged and uncharged phospholipids. As a result of the carrier conformational change, the ATPase complex is induced to prefer a negatively charged phospholipid environment.     

Critical role of the beta-subunit CDC50A in the stable expression, assembly, subcellular localization, and lipid transport activity of the P4-ATPase ATP8A2

P(4)-ATPases have been implicated in the transport of lipids across cellular membranes. Some P(4)-ATPases are known to associate with members of the CDC50 protein family. Previously, we have shown that the P(4)-ATPase ATP8A2 purified from photoreceptor membranes and reconstituted into liposomes catalyzes the active transport of phosphatidylserine across membranes. However, it was unclear whether ATP8A2 functioned alone or as a complex with a CDC50 protein. Here, we show by mass spectrometry and Western blotting using newly generated anti-CDC50A antibodies that CDC50A is associated with ATP8A2 purified from photoreceptor membranes. ATP8A2 expressed in HEK293T cells assembles with endogenous or expressed CDC50A, but not CDC50B, to generate a heteromeric complex that actively transports phosphatidylserine and to a lesser extent phosphatidylethanolamine across membranes. Chimera CDC50 proteins in which various domains of CDC50B were replaced with the corresponding domains of CDC50A were used to identify domains important in the formation of a functional ATP8A2-CDC50 complex. These studies indicate that both the transmembrane and exocytoplasmic domains of CDC50A are required to generate a functionally active complex. The N-terminal cytoplasmic domain of CDC50A appears to play a direct role in the reaction cycle. Mutagenesis studies further indicate that the N-linked oligosaccharide chains of CDC50A are required for stable expression of an active ATP8A2-CDC50A lipid transport complex. Together, our studies indicate that CDC50A is the β-subunit of ATP8A2 and is crucial for the correct folding, stable expression, export from endoplasmic reticulum, and phosphatidylserine flippase activity of ATP8A2.     

Tomato annexins p34 and p35 bind to F-actin and display nucleotide phosphodiesterase activity inhibited by phospholipid binding.

Annexins are a family of proteins found in a range of eukaryotic cell types. They share a characteristic amino acid sequence and a Ca(2+)-dependent affinity for specific phospholipids. In plants, proteins with common properties and significant homology with annexins have been identified in a number of species and implicated in diverse cellular functions known to be modulated by Ca2+. This study describes several novel biochemical properties of the tomato annexins p34 and p35 that are relevant to our understanding of their functions in the plant. First, the annexins were found to bind to actin in a calcium- and pH-dependent interaction that was specific for F-actin and not G-actin. Second, an enzyme activity defined as a nucleotide phosphodiesterase activity was found associated with the purified annexin preparation. Selective immunoprecipitation of p34 and p35 strongly suggests that the enzyme activity is a property of the annexins and constitutes 60% of the total soluble activity found in root extracts capable of hydrolyzing free ATP. The substrate specificity of the enzyme within in vitro assays is broad. ATP is the preferred substrate, but nearly identical rates of hydrolysis of GTP and substantial hydrolysis of other nucleotide tri- and diphosphates are observed. The enzyme activity was found to be a property of both p34 and p35, although the specific activity was routinely higher for p34. Third, the enzyme activity of the annexins was not affected by F-actin binding but could be abolished by the specific Ca(2+)-dependent interaction of the annexins with phospholipids. Our results showed that p34 and p35 account for substantial enzyme activity in tomato root cells. This activity was exhibited when the proteins were either in soluble form or attached to actin filaments. Enzyme activity was not exhibited when the annexins were bound to phospholipids. These properties suggest a role for the proteins in mediating Ca(2+)-dependent events involving interactions of the cytoskeleton and cellular membranes.     

The characterization of phospholipids associated with microtubules, purified tubulin and microtubule associated proteins in vitro

1. Significant levels of total phospholipid phosphate were detected in highly purified microtubule protein preparations. 2. While the phospholipid profiles of total microtubule proteins and microtubule-associated proteins showed both similarities and differences to that of a whole brain homogenate, purified tubulin was associated only with phospholipids that were not detectable in the latter. 3. Phosphatidyl ethanolamine, found exclusively in a fraction of microtubule associated proteins, stimulated microtubule assembly in vitro.     

Lipid environment of gastric potassium ion-stimulated adenosine triphosphatase.

The K+-stimulated ATPase associated with the purified gastric microsomal fraction can be completely inactivated by treatment with 15% (v/v) ethanol for 60s at 37 degrees C, but not at 25 degrees C. Sequential exposure of the microsomal fraction to 15% ethanol at 25 degrees C and 37 degrees C caused release of 2.5% and 2.9% of the total membrane phospholipids respectively. Restoration of the enzyme activity was achieved by sonication with phosphatidylcholine in the presence of Mg2+, K+ and ATP, which were essential for the reconstitution. Our data suggest that the phospholipids extracted by 15% ethanol at 37 degrees C are derived primarily from the immediate lipid environment of the enzyme, and ATP, together with the metal ions, helps the partially delipidated enzyme to retain the appropriate configuration for the subsequent reconstitution.     

EHD proteins are associated with tubular and vesicular compartments and interact with specific phospholipids

The four Eps15 homology (EH) domain-containing proteins, EHD1-EHD4, have recently been ascribed roles in the regulation of the recycling of distinct receptor molecules and are often found associated with tubular structures. Here, we report the analysis of all four EHD proteins with regard to tissue distribution, intracellular localization and lipid binding properties. Specific antibodies reveal distinct expression profiles for the individual proteins in tissues and at intracellular locations, where they potentially interact with specific phospholipids. Moreover, EHD proteins colocalize with vesicular and tubular structures, implying roles in transport processes and cytoskeletal dynamics. Protein variants carrying mutations in the N-terminal nucleotide-binding P-loop region are no longer associated with phospholipids or membrane compartments, while deletion of the C-terminal EH domain affects targeting to tubular structures. All EHD proteins are able to bind to phospholipids, but localizations differ for each protein.     

Facilitation of dissociation reaction of nucleotides bound to Mycobacterium tuberculosis DnaA

Acidic phospholipids have been shown to promote dissociation of bound nucleotides from Mycobacterium tuberculosis DnaA (DnaA(TB)) purified under denaturing conditions [Yamamoto et al., (2002) Modulation of Mycobacterium tuberculosis DnaA protein-adenine-nucleotide interactions by acidic phospholipids. Biochem. J., 363, 305-311]. In the present study, we show that a majority of DnaA(TB) in non-overproducing cells of M. tuberculosis is membrane associated. Estimation of phospholipid phosphorus following chloroform: methanol extraction of soluble DnaA(TB) purified under native conditions (nDnaA(TB)) confirmed the association with phospholipids. nDnaA(TB) exhibited weak ATPase activity, and rapidly exchanged ATP for bound ADP in the absence of any added phospholipids. We suggest that the outcome of intra-cellular DnaA(TB)-nucleotide interactions, hence DnaA(TB) activity, is influenced by phospholipids.     

Identification of novel phospholipid binding proteins in Saccharomyces cerevisiae

A proteomics approach was used to search for novel phospholipid binding proteins in Saccharomyces cerevisiae. Phospholipids were immobilized on a solid support and the lipids were probed with soluble yeast protein extracts. From this, the phosphatidic acid binding proteins were eluted and identified by mass spectrometry. Thirteen proteins were identified and 11 of these were previously unknown lipid binding proteins. The protein-lipid interactions identified would not have been predicted using bioinformatics approaches as none possessed a known lipid binding motif. A subset of the identified proteins was purified to homogeneity and determined to directly bind phospholipids immobilized on a solid support or organized into liposomes. This simple approach could be systematically applied to perform an exhaustive screen for soluble lipid binding proteins in S. cerevisiae or other organisms.     

Properties of a reconstituted calcium pump.

1. The translocation of 45Ca2+ in vesicles reconstituted with purified Ca2+ ATPase of sarcoplasmic reticulum and phospholipids was dependent on ATP and varied greatly with the composition of the phospholipids. 2. In contrast to sarcoplasmic reticulum fragments, the reconstituted vesicles were impermeable to 14C-labeled oxalate, 3H- or 32P-labeled ATP, or 32P-i. There was no translocation of phosphate from gamma-labeled ATP during Ca2+ uptake. These results are inconsistent with some current formulations of the mechanism of pump action. 3. Reversal of the Ca2+ pump and generation of ATP and ADP and P-i was observed when vesicles loaded with Ca2+ were exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 4. Experiments on the formation of phosphoenzyme with 32P-labeled ATP showed that most if not all functional ATPase molecules in the reconstituted vesicles were oriented in the same direction, as in the case of sarcoplasmic reticulum fragments.     

Heteromeric interactions required for abundance and subcellular localization of human CDC50 proteins and class 1 P4-ATPases

Members of the P(4) family of P-type ATPases (P(4)-ATPases) are believed to function as phospholipid flippases in complex with CDC50 proteins. Mutations in the human class 1 P(4)-ATPase gene ATP8B1 cause a severe syndrome characterized by impaired bile flow (intrahepatic cholestasis), often leading to end-stage liver failure in childhood. In this study, we determined the specificity of human class 1 P(4)-ATPase interactions with CDC50 proteins and the functional consequences of these interactions on protein abundance and localization of both protein classes. ATP8B1 and ATP8B2 co-immunoprecipitated with CDC50A and CDC50B, whereas ATP8B4, ATP8A1, and ATP8A2 associated only with CDC50A. ATP8B1 shifted from the endoplasmic reticulum (ER) to the plasma membrane upon coexpression of CDC50A or CDC50B. ATP8A1 and ATP8A2 translocated from the ER to the Golgi complex and plasma membrane upon coexpression of CDC50A, but not CDC50B. ATP8B2 and ATP8B4 already displayed partial plasma membrane localization in the absence of CDC50 coexpression but displayed a large increase in plasma membrane abundance upon coexpression of CDC50A. ATP8B3 did not bind CDC50A and CDC50B and was invariably present in the ER. Our data show that interactions between CDC50 proteins and class 1 P(4)-ATPases are essential for ER exit and stability of both subunits. Furthermore, the subcellular localization of the complex is determined by the P(4)-ATPase, not the CDC50 protein. The interactions of CDC50A and CDC50B with multiple members of the human P(4)-ATPase family suggest that these proteins perform broader functions in human physiology than thus far assumed.     

Phospholipid-protein interactions in the Ca2+-adenosine triphosphatase of sarcoplasmic reticulum.

Ca2+-adenosine triphosphatase from sarcoplasmic reticulum has been delipidated by gel filtration through a Sephadex G-200 column equilibrated with buffer containing cholate. The delipidated Ca2+-adenosine triphosphatase had negligible adenosine triphosphatase activity, but up to 50% of the ATPase activity was restored when the delipidated enzyme was recombined with phosphilipids. It was shown with the delipidated preparation that the phosphorylation of the enzyme by either ATP or Pi was entirely dependent on phospholipids. Among the purified phospholipids, phosphatidylcholine reactivated the adenosine triphosphatase activity better than phosphatidylethanolamine. Vesicles capable of translocating Ca2+ were reconstituted from delipidated Ca2+-adenosine triphosphatase and phosphatidylethanolamine, but not with phosphatidylcholine alone. We conclude that the firmly bound phospholipids which are purified together with the adenosine triphosphatase protein are not essential for the pump since they can be substituted by phosphatidylethanolamine isolated from soybeans.     

Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.     

Choline head groups stabilize the matrix loop regions of the ATP/ADP carrier ScAAC2

ATP/ADP carriers (AACs) are essential to the cell as they exchange ATP produced in mitochondria for cytosolic ADP. Monoclonal antibodies against the isoform 2 of Saccharomyces cerevisiae AAC (ScAAC2) were used to probe the accessibility of the matrix loops 1 and 3 depending on the environment of the carrier. In mitochondrial membranes ScAAC2 was not recognized, whereas in dodecylmaltoside the antibodies bound to the carrier, suggesting that the epitopes are hidden in the native environment. Exposure of the epitopes by detergents was reversed by reconstitution of the carrier in phospholipids or by exchanging with detergents having a choline or a trimethylammonium head group. Circular dichroism spectroscopy on peptides representing the C-terminal regions of all three matrix loops showed that only phosphocholine detergents induced a structural reorganization. Since in addition phosphatidylcholine was found to be tightly associated with the purified carrier, the matrix loop regions are likely to be associated to the membrane by phosphatidylcholine.     

A mutation (R826W) in nucleotide-binding domain 1 of ABCC8 reduces ATPase activity and causes transient neonatal diabetes

Activating mutations in the pore-forming Kir6.2 (KCNJ11) and regulatory sulphonylurea receptor SUR1 (ABCC8) subunits of the K(ATP) channel are a common cause of transient neonatal diabetes mellitus (TNDM). We identified a new TNDM mutation (R826W) in the first nucleotide-binding domain (NBD1) of SUR1. The mutation was found in a region that heterodimerizes with NBD2 to form catalytic site 2. Functional analysis showed that this mutation decreases MgATP hydrolysis by purified maltose-binding protein MBP-NBD1 fusion proteins. Inhibition of ATP hydrolysis by MgADP or BeF was not changed. The results indicate that the ATPase cycle lingers in the post-hydrolytic MgADP.P(i)-bound state, which is associated with channel activation. The extent of MgADP-dependent activation of K(ATP) channel activity was unaffected by the R826W mutation, but the time course of deactivation was slowed. Channel inhibition by MgATP was reduced, leading to an increase in resting whole-cell currents. In pancreatic beta cells, this would lead to less insulin secretion and thereby diabetes.     

Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes

A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADP-ribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mm nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mm ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, cAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20–200 μm) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.     

Immunolocalization of myosin I heavy chain kinase in Acanthamoeba castellanii and binding of purified kinase to isolated plasma membranes

The actin-activated Mg(2+)-ATPase activities of Acanthamoeba myosins I are known to be maximally expressed only when a single threonine (myosin IA) or serine (myosins IB and IC) is phosphorylated by myosin I heavy chain kinase. The purified kinase is highly activated by autophosphorylation and the rate of autophosphorylation is greatly enhanced by the presence of acidic phospholipids. In this paper, we show by immunofluorescence and immunoelectron microscopy of permeabilized cells that myosin I heavy chain kinase is highly concentrated, but not exclusively, at the plasma membrane. Judged by their electrophoretic mobilities, kinase associated with purified plasma membranes may differ from the cytoplasmic kinase, possibly in the extent of its phosphorylation. Purified kinase binds to highly purified plasma membranes with an apparent KD of approximately 17 nM and a capacity of approximately 0.8 nmol/mg of plasma membrane protein, values that are similar to the affinity and capacity of plasma membranes for myosins I. Binding of kinase to membranes is inhibited by elevated ionic strength and by extensive autophosphorylation but not by substrate-level concentrations of ATP. Membrane-bound kinase autophosphorylates to a lesser extent than free kinase and does not dissociate from the membranes after autophosphorylation. The co-localization of myosin I heavy chain kinase and myosin I at the plasma membrane is of interest in relation to the possible functions of myosin I especially as phospholipids increase kinase activity.     

On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids

The activation of purified and phospholipid-depleted plasma membrane Ca2+-ATPase by phospholipids and ATP was studied. Enzyme activity increased with [ATP] along biphasic curves representing the sum of two Michaelis-Menten equations. Acidic phospholipids (phosphatidylinositol (PI) and phosphatidylserine (PS)) increased Vmax without affecting apparent affinities of the ATP sites. In the presence of 20 microm ATP, phosphorylation of the enzyme preincubated with Ca2+ (CaE1) was very fast (kapp congruent with 400 s-1). vo of phosphorylation of CaE1 increased with [ATP] along a Michaelis-Menten curve (Km of 15 microm) and was phospholipid-independent. Without Ca2+ preincubation (E1 + E2), vo of phosphorylation was also phospholipid-independent, but was slower and increased with [ATP] along biphasic curves. The high affinity component reflected rapid phosphorylation of CaE1, the low affinity component the E2 --> E1 shift, which accelerated to a rate higher than that of the ATPase activity when ATP was bound to the regulatory site. Dephosphorylation of EP did not occur without ATP. Dephosphorylation increased along a biphasic curve with increasing [ATP], showing that ATP accelerated dephosphorylation independently of phospholipid. PI, but not phosphatidylethanolamine (PE), accelerated dephosphorylation even in the absence of ATP. kapp for dephosphorylation was 57 s-1 at 0 microM ATP; that rate was further increased by ATP. Steady-state [EP] x kapp for dephosphorylation varied with [ATP], and matched the Ca2+-ATPase activity measured under the same conditions. Apparently, the catalytic cycle is rate-limited by dephosphorylation. Acidic phospholipids stimulate Ca2+-ATPase activity by accelerating dephosphorylation, while ATP accelerates both dephosphorylation and the conformational change from E2 to E1, further stimulating the ATPase activity.