Selective Transport of α-Mannosidase by Autophagic Pathways: IDENTIFICATION OF A NOVEL RECEPTOR,Atg34p*

In Saccharomyces cerevisiae, aminopeptidase I (Ape1p) and α-mannosidase (Ams1p) are known cargoes of selective autophagy. Atg19p has been identified as an Ape1p receptor and targets Ape1p to the preautophagosomal structure (PAS). Under nutrient-rich conditions, transport of Ams1p to the vacuole largely depends on Atg19p. Here, we show that Atg34p (Yol083wp), a homolog of Atg19p, is a receptor for Ams1p transport during autophagy. Atg34p interacted with Ams1p, Atg11p, and Atg8p using distinct domains. Homo-oligomerized Ams1p bound to the Ams1-binding domain of Atg34p; this binding was important for the formation of a higher order complex named the Ams1 complex. In the absence of the interaction of Atg34p with Atg8p, the Ams1 complex was targeted to the preautophagosomal structure but failed to transit to the vacuole, indicating that the interaction of Atg34p with Atg8p is crucial for the Ams1 complex to be enclosed by autophagosomes. Atg34p and Atg19p have similar domain structures and are important for Ams1p transport during autophagy.     

Complexes of Trypanosoma cruzi Sterol 14α-Demethylase (CYP51) with Two Pyridine-based Drug Candidates for Chagas Disease: STRUCTURAL BASIS FOR PATHOGEN SELECTIVITY*

Chagas disease, caused by the eukaryotic (protozoan) parasite Trypanosoma cruzi, is an alarming emerging global health problem with no clinical drugs available to treat the chronic stage. Azole inhibitors of sterol 14α-demethylase (CYP51) were proven effective against Chagas, and antifungal drugs posaconazole and ravuconazole have entered clinical trials in Spain, Bolivia, and Argentina. Here we present the x-ray structures of T. cruzi CYP51 in complexes with two alternative drug candidates, pyridine derivatives (S)-(4-chlorophenyl)-1-(4-(4-(trifluoromethyl)phenyl)-piperazin-1-yl)-2-(pyridin-3-yl)ethanone (UDO; Protein Data Bank code 3ZG2) and N-[4-(trifluoromethyl)phenyl]-N-[1-[5-(trifluoromethyl)-2-pyridyl]-4-piperi-dyl]pyridin-3-amine (UDD; Protein Data Bank code 3ZG3). These compounds have been developed by the Drugs for Neglected Diseases initiative (DNDi) and are highly promising antichagasic agents in both cellular and in vivo experiments. The binding parameters and inhibitory effects on sterol 14α-demethylase activity in reconstituted enzyme reactions confirmed UDO and UDD as potent and selective T. cruzi CYP51 inhibitors. Comparative analysis of the pyridine- and azole-bound CYP51 structures uncovered the features that make UDO and UDD T. cruzi CYP51-specific. The structures suggest that although a precise fit between the shape of the inhibitor molecules and T. cruzi CYP51 active site topology underlies their high inhibitory potency, a longer coordination bond between the catalytic heme iron and the pyridine nitrogen implies a weaker influence of pyridines on the iron reduction potential, which may be the basis for the observed selectivity of these compounds toward the target enzyme versus other cytochrome P450s, including human drug-metabolizing P450s. These findings may pave the way for the development of novel CYP51-targeted drugs with optimized metabolic properties that are very much needed for the treatment of human infections caused by eukaryotic microbial pathogens.     

The Power of Two: ARGININE 51 AND ARGININE 239* FROM A NEIGHBORING SUBUNIT ARE ESSENTIAL FOR CATALYSIS IN α-AMINO-β-CARBOXYMUCONATE-?-SEMIALDEHYDE DECARBOXYLASE*

Although the crystal structure of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase from Pseudomonas fluorescens was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 Å, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 Å resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P4₂2₁2 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wild-type structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β-carboxymuconate-ϵ-semialdehyde decarboxylase could regulate the enzyme activity.     

Trafficking of α4* Nicotinic Receptors Revealed by Superecliptic Phluorin: EFFECTS OF A β4 AMYOTROPHIC LATERAL SCLEROSIS-ASSOCIATED MUTATION AND CHRONIC EXPOSURE TO NICOTINE*

We employed a pH-sensitive GFP analog, superecliptic phluorin, to observe aspects of nicotinic acetylcholine receptor (nAChR) trafficking to the plasma membrane (PM) in cultured mouse cortical neurons. The experiments exploit differences in the pH among endoplasmic reticulum (ER), trafficking vesicles, and the extracellular solution. The data confirm that few α4β4 nAChRs, but many α4β2 nAChRs, remain in neutral intracellular compartments, mostly the ER. We observed fusion events between nAChR-containing vesicles and PM; these could be quantified in the dendritic processes. We also studied the β4R348C polymorphism, linked to amyotrophic lateral sclerosis (ALS). This mutation depressed fusion rates of α4β4 receptor-containing vesicles with the PM by ∼2-fold, with only a small decrease in the number of nAChRs per vesicle. The mutation also decreased the number of ER exit sites, showing that the reduced receptor insertion results from a change at an early stage in trafficking. We confirm the previous report that the mutation leads to reduced agonist-induced currents; in the cortical neurons studied, the reduction amounts to 2–3-fold. Therefore, the reduced agonist-induced currents are caused by the reduced number of α4β4-containing vesicles reaching the membrane. Chronic nicotine exposure (0.2 μm) did not alter the PM insertion frequency or trafficking behavior of α4β4-laden vesicles. In contrast, chronic nicotine substantially increased the number of α4β2-containing vesicle fusions at the PM; this stage in α4β2 nAChR up-regulation is presumably downstream from increased ER exit. Superecliptic phluorin provides a tool to monitor trafficking dynamics of nAChRs in disease and addiction.     

Characterization of Semisynthetic and Naturally Nα-Acetylated α-Synuclein in Vitro and in Intact Cells: IMPLICATIONS FOR AGGREGATION AND CELLULAR PROPERTIES OF α-SYNUCLEIN

N-terminal acetylation is a very common post-translational modification, although its role in regulating protein physical properties and function remains poorly understood. α-Synuclein (α-syn), a protein that has been linked to the pathogenesis of Parkinson disease, is constitutively N(α)-acetylated in vivo. Nevertheless, most of the biochemical and biophysical studies on the structure, aggregation, and function of α-syn in vitro utilize recombinant α-syn from Escherichia coli, which is not N-terminally acetylated. To elucidate the effect of N(α)-acetylation on the biophysical and biological properties of α-syn, we produced N(α)-acetylated α-syn first using a semisynthetic methodology based on expressed protein ligation (Berrade, L., and Camarero, J. A. (2009) Cell. Mol. Life Sci. 66, 3909-3922) and then a recombinant expression strategy, to compare its properties to unacetylated α-syn. We demonstrate that both WT and N(α)-acetylated α-syn share a similar secondary structure and oligomeric state using both purified protein preparations and in-cell NMR on E. coli overexpressing N(α)-acetylated α-syn. The two proteins have very close aggregation propensities as shown by thioflavin T binding and sedimentation assays. Furthermore, both N(α)-acetylated and WT α-syn exhibited similar ability to bind synaptosomal membranes in vitro and in HeLa cells, where both internalized proteins exhibited prominent cytosolic subcellular distribution. We then determined the effect of attenuating N(α)-acetylation in living cells, first by using a nonacetylable mutant and then by silencing the enzyme responsible for α-syn N(α)-acetylation. Both approaches revealed similar subcellular distribution and membrane binding for both the nonacetylable mutant and WT α-syn, suggesting that N-terminal acetylation does not significantly affect its structure in vitro and in intact cells.     

Diversity of Structure and Function of α1α6β3δ GABAA Receptors: COMPARISON WITH α1β3δ AND α6β3δ RECEPTORS*

δ subunit-containing γ-aminobutyric acid, type A (GABA_A)receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with α₁ and/or α₆ subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA_A receptor pentamers by concatenation. These receptors composed of α₁, α₆, β₃, and δ subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3α,21-dihydroxy-5α-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that δ can assume multiple positions in a receptor pentamer made up of α₁, α₆, β₃, and δ subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of δ subunits between two α subunits in α₁α₆β₃δ receptors. This property is shared by α₁β₃δ and α₆β₃δ receptors, but there are differences in the additionally expressed isoforms.     

Determination of α2HS-glycoprotein phenotypes by isoelectric focusing and immunoblotting: polymorphic occurrence of HSGA *5 in Okinawa

Summary A print immunofixation is a very useful procedure for the demonstration of ₂HS-glycoprotein (HSGA) following polyacrylamide gel isoelectric focusing. However, this technique has the one disadvantage of requiring a large volume of expensive antiserum. In this paper an alternative detection system is presented which involves the non-electric transfer of HSGA from a focused gel to a nitrocellulose filter and the immunologic detection of HSGA immobilized on nitrocellulose. Using this method the distribution of HSGA polymorphism in the Nepalese and Japanese populations was investigated. All the populations tested were found to lack the common HSGA ^*3 of the Caucasians. A rare HSGA ^*5 in Honshu, a main island of Japan, was observed at polymorphic frequency in Okinawa, Southern Japan.     

Cell Wall β-(1,6)-Glucan of Saccharomyces cerevisiae: STRUCTURAL CHARACTERIZATION AND IN SITU SYNTHESIS*S?

Despite its essential role in the yeast cell wall, the exact composition of the β-(1,6)-glucan component is not well characterized. While solubilizing the cell wall alkali-insoluble fraction from a wild type strain of Saccharomyces cerevisiae using a recombinant β-(1,3)-glucanase followed by chromatographic characterization of the digest on an anion exchange column, we observed a soluble polymer that eluted at the end of the solvent gradient run. Further characterization indicated this soluble polymer to have a molecular mass of ∼38 kDa and could be hydrolyzed only by β-(1,6)-glucanase. Gas chromatographymass spectrometry and NMR (¹H and ¹³C) analyses confirmed it to be a β-(1,6)-glucan polymer with, on average, branching at every fifth residue with one or two β-(1,3)-linked glucose units in the side chain. This polymer peak was significantly reduced in the corresponding digests from mutants of the kre genes (kre9 and kre5) that are known to play a crucial role in the β-(1,6)-glucan biosynthesis. In the current study, we have developed a biochemical assay wherein incubation of UDP-[¹⁴C]glucose with permeabilized S. cerevisiae yeasts resulted in the synthesis of a polymer chemically identical to the branched β-(1,6)-glucan isolated from the cell wall. Using this assay, parameters essential for β-(1,6)-glucan synthetic activity were defined.The cell wall of Saccharomyces cerevisiae and other yeasts contains two types of β-glucans. In the former yeast, branched β-(1,3)-glucan accounts for ∼50–55%, whereas β-(1,6)-glucan represents 10–15% of the total yeast cell wall polysaccharides, each chain of the latter extending up to 140–350 glucose residues in length. The amount of 3,6-branched glucose residues varies with the yeast species: 7, 15, and 75% in S. cerevisiae, Candida albicans, and Schizosaccharomyces pombe, respectively (1). β-(1,6)-Glucan stabilizes the cell wall, since it plays a central role as a linker for specific cell wall components, including β-(1,3)-glucan, chitin, and mannoproteins (2, 3). However, the exact structure of the β-(1,6)-glucan and the mode of biosynthesis of this polymer are largely unknown. In S. pombe, immunodetection studies suggested that synthesis of this polymer backbone begins in the endoplasmic reticulum, with extension occurring in the Golgi (4) and final processing at the plasma membrane. In S. cerevisiae, Montijn and co-workers (5), by immunogold labeling, detected β-(1,6)-glucan at the plasma membrane, suggesting that the synthesis takes place largely at the cell surface.More than 20 genes, including the KRE gene family (14 members) and their homologues, SKN1 and KNH1, have been reported to be involved in β-(1,6)-glucan synthesis in S. cerevisiae, C. albicans, and Candida glabrata (6–10). Among all of these genes, the ones that seem to play the major synthetic role are KRE5 and KRE9, since their disruption caused significant reduction (100 and 80%, respectively, relative to wild type) in the cell wall β-(1,6)-glucan content (11–13).To date, the biochemical reaction responsible for the synthesis of β-(1,6)-glucan and the product synthesized remained unknown. Indeed, in most cases, when membrane preparations are incubated with UDP-glucose, only linear β-(1,3)-glucan polymers are produced, although some studies have reported the production of low amounts of β-(1,6)-glucans by membrane preparations (14–17). These data suggest that disruption of the fungal cell prevents or at least has a strong negative effect on β-(1,6)-glucan synthesis. The use of permeabilized cells, which allows substrates, such as nucleotide sugar precursors, to be readily transported across the plasma membrane, is an alternative method to study in situ cell wall enzyme activities (18–22). A number of methods have been developed to permeabilize the yeast cell wall (23), of which osmotic shock was successfully used to demonstrate β-(1,3)-glucan and chitin synthase activities (20, 24). Herein, we describe the biochemical activity responsible for β-(1,6)-glucan synthesis using permeabilized S. cerevisiae cells and UDP-[¹⁴C]glucose as a substrate. We also have analyzed the physicochemical parameters of this activity and chemically characterized the end product and its structural organization within the mature yeast cell wall.     

Enzymatic Basis for N-Glycan Sialylation: STRUCTURE OF RAT α2,6-SIALYLTRANSFERASE (ST6GAL1) REVEALS CONSERVED AND UNIQUE FEATURES FOR GLYCAN SIALYLATION*

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.     

Assembly of the Bak Apoptotic Pore: A CRITICAL ROLE FOR THE BAK PROTEIN α6 HELIX IN THE MULTIMERIZATION OF HOMODIMERS DURING APOPTOSIS*

Bak and Bax are the essential effectors of the intrinsic pathway of apoptosis. Following an apoptotic stimulus, both undergo significant changes in conformation that facilitates their self-association to form pores in the mitochondrial outer membrane. However, the molecular structures of Bak and Bax oligomeric pores remain elusive. To characterize how Bak forms pores during apoptosis, we investigated its oligomerization under native conditions using blue native PAGE. We report that, in a healthy cell, inactive Bak is either monomeric or in a large complex involving VDAC2. Following an apoptotic stimulus, activated Bak forms BH3:groove homodimers that represent the basic stable oligomeric unit. These dimers multimerize to higher-order oligomers via a labile interface independent of both the BH3 domain and groove. Linkage of the α6:α6 interface is sufficient to stabilize higher-order Bak oligomers on native PAGE, suggesting an important role in the Bak oligomeric pore. Mutagenesis of the α6 helix disrupted apoptotic function because a chimera of Bak with the α6 derived from Bcl-2 could be activated by truncated Bid (tBid) and could form BH3:groove homodimers but could not form high molecular weight oligomers or mediate cell death. An α6 peptide could block Bak function but did so upstream of dimerization, potentially implicating α6 as a site for activation by BH3-only proteins. Our examination of native Bak oligomers indicates that the Bak apoptotic pore forms by the multimerization of BH3:groove homodimers and reveals that Bak α6 is not only important for Bak oligomerization and function but may also be involved in how Bak is activated by BH3-only proteins.     

Evolution of Substrate Specificity within a Diverse Family of β/α-Barrel-fold Basic Amino Acid Decarboxylases: X-RAY STRUCTURE DETERMINATION OF ENZYMES WITH SPECIFICITY FOR l-ARGININE AND CARBOXYNORSPERMIDINE*

Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu³¹⁴, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.     

Interaction of the Human Prostacyclin Receptor with Rab11: CHARACTERIZATION OF A NOVEL Rab11 BINDING DOMAIN WITHIN α-HELIX 8 THAT IS REGULATED BY PALMITOYLATION*

The human prostacyclin receptor (hIP) undergoes agonist-induced internalization and subsequent recyclization in slowly recycling endosomes involving its direct physical interaction with Rab11a. Moreover, interaction with Rab11a localizes to a 22-residue putative Rab11 binding domain (RBD) within the carboxyl-terminal tail of the hIP, proximal to the transmembrane 7 (TM7) domain. Because the proposed RBD contains Cys³⁰⁸ and Cys³¹¹, in addition to Cys³⁰⁹, that are known to undergo palmitoylation, we sought to identify the structure/function determinants of the RBD, including the influence of palmitoylation, on agonist-induced trafficking of the hIP. Through complementary approaches in yeast and mammalian cells along with computational structural studies, the RBD was localized to a 14-residue domain, between Val²⁹⁹ and Leu³¹², and proposed to be organized into an eighth α-helical domain (α-helix 8), comprising Val²⁹⁹–Val³⁰⁷, adjacent to the palmitoylated residues at Cys³⁰⁸–Cys³¹¹. From mutational and [³H]palmitate metabolic labeling studies, it is proposed that palmitoylation at Cys³¹¹ in addition to agonist-regulated deacylation at Cys³⁰⁹ > Cys³⁰⁸ may dynamically position α-helix 8 in proximity to Rab11a, to regulate agonist-induced intracellular trafficking of the hIP. Moreover, Ala-scanning mutagenesis identified several hydrophobic residues within α-helix 8 as necessary for the interaction with Rab11a. Given the diverse membership of the G protein-coupled receptor superfamily, of which many members are also predicted to contain an α-helical 8 domain proximal to TM7 and, often, adjacent to palmitoylable cysteine(s), the identification of a functional role for α-helix 8, as exemplified as an RBD for the hIP, is likely to have broader significance for certain members of the superfamily.     

Structure,Folding Dynamics,and Amyloidogenesis of D76N β2-Microglobulin: ROLES OF SHEAR FLOW,HYDROPHOBIC SURFACES,AND α-CRYSTALLIN*

Systemic amyloidosis is a fatal disease caused by misfolding of native globular proteins, which then aggregate extracellularly as insoluble fibrils, damaging the structure and function of affected organs. The formation of amyloid fibrils in vivo is poorly understood. We recently identified the first naturally occurring structural variant, D76N, of human β₂-microglobulin (β₂m), the ubiquitous light chain of class I major histocompatibility antigens, as the amyloid fibril protein in a family with a new phenotype of late onset fatal hereditary systemic amyloidosis. Here we show that, uniquely, D76N β₂m readily forms amyloid fibrils in vitro under physiological extracellular conditions. The globular native fold transition to the fibrillar state is primed by exposure to a hydrophobic-hydrophilic interface under physiological intensity shear flow. Wild type β₂m is recruited by the variant into amyloid fibrils in vitro but is absent from amyloid deposited in vivo. This may be because, as we show here, such recruitment is inhibited by chaperone activity. Our results suggest general mechanistic principles of in vivo amyloid fibrillogenesis by globular proteins, a previously obscure process. Elucidation of this crucial causative event in clinical amyloidosis should also help to explain the hitherto mysterious timing and location of amyloid deposition.     

Regulation of Autophagy and Its Associated Cell Death by “Sphingolipid Rheostat”: RECIPROCAL ROLE OF CERAMIDE AND SPHINGOSINE 1-PHOSPHATE IN THE MAMMALIAN TARGET OF RAPAMYCIN PATHWAY*

The role of “sphingolipid rheostat” by ceramide and sphingosine 1-phosphate (S1P) in the regulation of autophagy remains unclear. In human leukemia HL-60 cells, amino acid deprivation (AA(−)) caused autophagy with an increase in acid sphingomyleinase (SMase) activity and ceramide, which serves as an autophagy inducing lipid. Knockdown of acid SMase significantly suppressed the autophagy induction. S1P treatment counteracted autophagy induction by AA(−) or C₂-ceramide. AA(−) treatment promoted mammalian target of rapamycin (mTOR) dephosphorylation/inactivation, inducing autophagy. S1P treatment suppressed mTOR inactivation and autophagy induction by AA(−). S1P exerts biological actions via cell surface receptors, and S1P₃ among five S1P receptors was predominantly expressed in HL-60 cells. We evaluated the involvement of S1P₃ in suppressing autophagy induction. S1P treatment of CHO cells had no effects on mTOR inactivation and autophagy induction by AA(−) or C₂-ceramide. Whereas S1P treatment of S1P₃ overexpressing CHO cells resulted in activation of the mTOR pathway, preventing cells from undergoing autophagy induced by AA(−) or C₂-ceramide. These results indicate that S1P-S1P₃ plays a role in counteracting ceramide signals that mediate mTOR-controlled autophagy. In addition, we evaluated the involvement of ceramide-activated protein phosphatases (CAPPs) in ceramide-dependent inactivation of the mTOR pathway. Inhibition of CAPP by okadaic acid in AA(−)- or C₂-ceramide-treated cells suppressed dephosphorylation/inactivation of mTOR, autophagy induction, and autophagy-associated cell death, indicating a novel role of ceramide-CAPPs in autophagy induction. Moreover, S1P₃ engagement by S1P counteracted cell death. Taken together, these results indicated that sphingolipid rheostat in ceramide-CAPPs and S1P-S1P₃ signaling modulates autophagy and its associated cell death through regulation of the mTOR pathway.