Xiao Song Xie

Cardiolipin provides specificity for targeting of tBid to mitochondria

Recent evidence supports the theory that mitochondrial homeostasis is the key regulatory step in apoptosis through the actions of members of the Bcl-2 family. Pro-apoptotic members of the family, such as Bax, Bad and Bid, can induce the loss of outer-membrane integrity with subsequent redistribution of pro-apoptotic proteins such as cytochrome c that are normally located in the intermembrane spaces of mitochondria. The anti-apoptotic members of the family, such as Bcl-2 and Bcl-XL, protect the integrity of the mitochondrion and prevent the release of death-inducing factors. Bid normally exists in an inactive state in the cytosol, but after cleavage by caspase 8, the carboxy-terminal portion (tBid) moves from cytosol to mitochondria, where it induces release of cytochrome c. Here we address the question of what mediates specific targeting of tBid to the mitochondria. We provide evidence that cardiolipin, which is present in mitochondrial membranes, mediates the targeting of tBid to mitochondria through a previously unkown three-helix domain in tBid. These findings implicate cardiolipin in the pathway for cytochrome c release.

C. elegans mitochondrial factor WAH-1 promotes phosphatidylserine externalization in apoptotic cells through phospholipid scramblase SCRM-1

Externalization of phosphatidylserine, which is normally restricted to the inner leaflet of plasma membrane, is a hallmark of mammalian apoptosis. It is not known what activates and mediates the phosphatidylserine externalization process in apoptotic cells. Here, we report the development of an annexin V-based phosphatidylserine labelling method and show that a majority of apoptotic germ cells in Caenorhabditis elegans have surface-exposed phosphatidylserine, indicating that phosphatidylserine externalization is a conserved apoptotic event in worms. Importantly, inactivation of the gene encoding either the C. elegans apoptosis-inducing factor (AIF) homologue (WAH-1), a mitochondrial apoptogenic factor, or the C. elegans phospholipid scramblase 1 (SCRM-1), a plasma membrane protein, reduces phosphatidylserine exposure on the surface of apoptotic germ cells and compromises cell-corpse engulfment. WAH-1 associates with SCRM-1 and activates its phospholipid scrambling activity in vitro. Thus WAH-1, after its release from mitochondria during apoptosis, promotes plasma membrane phosphatidylserine externalization through its downstream effector, SCRM-1.

Identification and Functional Expression of Four Isoforms of ATPase II, the Putative Aminophospholipid Translocase EFFECT OF ISOFORM VARIATION ON THE ATPase ACTIVITY AND PHOSPHOLIPID SPECIFICITY

ATPase II, a vanadate-sensitive and phosphatidylserine-dependent Mg2+-ATPase, is a member of a subfamily of P-type ATPase and is presumably responsible for aminophospholipid translocation activity in eukaryotic cells. The aminophospholipid translocation activity plays an important physiological role in the maintenance of membrane phospholipid asymmetry that is observed in the plasma membrane as well as the membranes of certain cellular organelles. While the preparations of ATPase II from different sources share common fundamental properties, such as substrate specificity, inhibitor spectrum, and phospholipid dependence, they are divergent in several characteristics. These include specific ATPase activity and phospholipid selectivity. We report here the identification of four isoforms of ATPase II in bovine brain. These isoforms are formed by a combination of two major variations in their primary sequences and show that the structural variation of these isoforms has functional significance in both ATPase activity and phosholipid selectivity. Furthermore, studies with the phosphoenzyme intermediate of ATPase II and its recombinant isoforms revealed that phosphatidylserine is essential for the dephosphorylation of the intermediate. Without phosphatidylserine, ATPase II would be accumulated as phosphoenzyme in the presence of ATP, resulting in the interruption of its catalytic cycle.

Sterol Transfer by ABCG5 and ABCG8 IN VITRO ASSAY AND RECONSTITUTION

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either half-transporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using “inside-out” membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5- and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP ≫ CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer.

Crystal structure of the human sterol transporter ABCG5/ABCG8.

ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines. Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.

Relative roles of ABCG5/ABCG8 in liver and intestine

ABCG5 (G5) and ABCG8 (G8) form a sterol transporter that acts in liver and intestine to prevent accumulation of dietary sterols. Mutations in either G5 or G8 cause sitosterolemia, a recessive disorder characterized by sterol accumulation and premature coronary atherosclerosis. Hepatic G5G8 mediates cholesterol excretion into bile, but the function and relative importance of intestinal G5G8 has not been defined. To determine the role of intestinal G5G8, we developed liver-specific (L-G5G8−/−), intestine-specific (I-G5G8−/−), and total (G5G8−/−) KO mice. Tissue levels of sitosterol, the most abundant plant sterol, were >90-fold higher in G5G8−/− mice than in WT animals. Expression of G5G8 only in intestine or only in liver decreased tissue sterol levels by 90% when compared with G5G8−/− animals. Biliary sterol secretion was reduced in L-G5G8−/− and G5G8−/− mice, but not in I-G5G8−/− mice. Conversely, absorption of plant sterols was increased in I-G5G8−/− and G5G8−/− mice, but not in L-G5G8−/− mice. Reverse cholesterol transport, as assessed from the fraction of intravenously administered 3H-cholesterol that appeared in feces, was reduced in G5G8−/−, I-G5G8−/−, and L-G5G8−/− mice. Thus, G5G8 expression in both the liver and intestine protects animals from sterol accumulation, and intestinal G5G8 contributes to extrahepatic cholesterol efflux in mice.

Vacuolar proton pumps

Recently a new class of proton-translocating ATPases has been localized to endomembrane compartments in plant, fungal, and mammalian cells. These proton pumps are large hetero-oligomers which have an ATP hydrolytic sector that is functionally and structurally distinct from a transmembranous proton pore. Enzymatic characteristics of these proton pumps are discussed as well as the current state of knowledge regarding subunit composition and function. In addition, recent primary sequence data are discussed which indicate that these proton pumps share a common ancestor with F1F0-type proton pumps of mitochondria

Characterization of the Functional Coupling of Bovine Brain Vacuolar-type H+-translocating ATPase EFFECT OF DIVALENT CATIONS, PHOSPHOLIPIDS, AND SUBUNIT H (SFD)

Vacuolar-type H+-translocating ATPases (V-ATPases or V-pumps) are complex proteins containing multiple subunits and are organized into two functional domains: a peripheral catalytic sector V1 and a membranous proton channel V0. The functional coupling of ATP hydrolysis activity to proton transport in V-pumps requires a regulatory component known as subunit H (SFD) as has been shown both in vivo and in vitro (Ho, M. N., Hirata, R., Umemoto, N., Ohya, Y., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1993) J. Biol. Chem. 268, 18286–18292; Xie, X. S., Crider, B. P., Ma, Y. M., and Stone, D. K. (1994) J. Biol. Chem. 269, 25809–25815). Ca2+ is thought to uncouple V-pumps because it is found to support ATP hydrolysis but not proton transport, while Mg2+ supports both activities. The direct effect of phospholipids on the coupling of V-ATPases has not been reported, likely due to the fact that phospholipids are constituents of biological membranes. We now report that Ca2+-induced uncoupling of the bovine brain V-ATPase can be reversed by imposition of a favorable membrane potential. Furthermore we report a simple “membrane-free” assay system using the V0 proton channel-specific inhibitor bafilomycin as a probe to detect the coupling of V-ATPase under certain conditions. With this system, we have characterized the functional effect of subunit H, divalent cations, and phospholipids on bovine brain V-ATPase and have found that each of these three factors plays a critical role in the functional coupling of the V-pump.

Purification and reconstitution of sterol transfer by native mouse ABCG5 and ABCG8.

ABCG5 (G5) and ABCG8 (G8) are ATP-binding cassette half-transporters that limit intestinal uptake and promote biliary secretion of neutral sterols. Here, we describe the purification of endogenous G5G8 from mouse liver to near homogeneity. We incorporated the native proteins into membrane vesicles and reconstituted sterol transfer. Native gel electrophoresis, density-gradient ultracentrifugation, and chemical cross-linking studies indicated that the functional native complex is a heterodimer. No higher order oligomeric forms were observed at any stage in the catalytic cycle. Sterol transfer activity by purified native G5G8 was stable, stereospecific, and selective. We also report that G5 but not G8 is S-palmitoylated and that palmitoylation is not essential for dimerization, trafficking, or biliary sterol secretion. Both G5 and G8 have short but highly conserved cytoplasmic tails. The functional roles of these C-terminal regions were examined using an in vivo functional assay.

Molecular Characterization of the 50- and 57-kDa Subunits of the Bovine Vacuolar Proton Pump

The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel. In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump. These proteins, termed SFD (sub-fifty-eight-kDadimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem. 269, 28509–25815). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592–31597). We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional. To determine the true molecular identity of SFD, both the 50- and 57-kDa polypeptides were directly sequenced. A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA library, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a reverse transcription-polymerase chain reaction-based strategy to isolate a related, but distinct, transcript present in bovine brain mRNA. The nucleotide and predicted amino acid sequences of this isolate are identical to SFD-21 except that the isolate contains a 54-base pair insert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da. Both clones had 16% identity toVMA13 of Saccharomyces cerevisiae. No sequence homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epitope common to the two proteins and to the unique 18-amino acid insert of the larger protein. The former reacted with both components of native SFD, whereas the latter reacted only with the 57-kDa component. We term the 57- and 50-kDa polypeptides SFDα and SFDβ, respectively.