DDHD1, but Not DDHD2, Suppresses Neurite Outgrowth in SH-SY5Y and PC12 Cells by Regulating Protein Transport From Recycling Endosomes.

DDHD1 and DDHD2 are both intracellular phospholipases A1 and hydrolyze phosphatidic acid in vitro. Given that phosphatidic acid participates in neurite outgrowth, we examined whether DDHD1 and DDHD2 regulate neurite outgrowth. Depletion of DDHD1 from SH-SY5Y and PC12 cells caused elongation of neurites, whereas DDHD2 depletion prevented neurite elongation. Rescue experiments demonstrated that the enzymatic activity of DDHD1 is necessary for the prevention of neurite elongation. Depletion of DDHD1 caused enlargement of early endosomes and stimulated tubulation of recycling endosomes positive for phosphatidic acid-binding proteins syndapin2 and MICAL-L1. Knockout of DDHD1 enhanced transferrin recycling from recycling endosomes to the cell surface. Our results suggest that DDHD1 negatively controls the formation of a local phosphatidic acid-rich domain in recycling endosomes that serves as a membrane source for neurite outgrowth.

Loss of DDHD2, whose mutation causes spastic paraplegia, promotes reactive oxygen species generation and apoptosis.

DDHD2/KIAA0725p is a mammalian intracellular phospholipase A1 that exhibits phospholipase and lipase activities. Mutation of the DDHD2 gene causes hereditary spastic paraplegia (SPG54), an inherited neurological disorder characterized by lower limb spasticity and weakness. Although previous studies demonstrated lipid droplet accumulation in the brains of SPG54 patients and DDHD2 knockout mice, the cause of SPG54 remains elusive. Here, we show that ablation of DDHD2 in mice induces age-dependent apoptosis of motor neurons in the spinal cord. In vitro, motor neurons and embryonic fibroblasts from DDHD2 knockout mice fail to survive and are susceptible to apoptotic stimuli. Chemical and probe-based analysis revealed a substantial decrease in cardiolipin content and an increase in reactive oxygen species generation in DDHD2 knockout cells. Reactive oxygen species production in DDHD2 knockout cells was reversed by the expression of wild-type DDHD2, but not by an active-site DDHD2 mutant, DDHD2 mutants related to hereditary spastic paraplegia, or DDHD1, another member of the intracellular phospholipase A1 family whose mutation also causes spastic paraplegia (SPG28). Our results demonstrate the protective role of DDHD2 for mitochondrial integrity and provide a clue to the pathogenic mechanism of SPG54.

The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based WAVE2 complex.

BACKGROUND: Abl interactor (Abi) family proteins play significant roles in actin cytoskeleton organization through participation in the WAVE complex. Mammals possess three Abi proteins: Abi-1, Abi-2, and NESH/Abi-3. Abi-1 and Abi-2 were originally identified as Abl tyrosine kinase-binding proteins. It has been disclosed that Abi-1 acts as a bridge between c-Abl and WAVE2, and c-Abl-mediated WAVE2 phosphorylation promotes actin remodeling. We showed previously that NESH/Abi-3 is present in the WAVE2 complex, but neither binds to c-Abl nor promotes c-Abl-mediated phosphorylation of WAVE2. RESULTS: In this study, we characterized NESH/Abi-3 in more detail, and compared its properties with those of Abi-1 and Abi-2. NESH/Abi-3 was ectopically expressed in NIH3T3 cells, in which Abi-1, but not NESH/Abi-3, is expressed. The expression of NESH/Abi-3 caused degradation of endogenous Abi-1, which led to the formation of a NESH/Abi-3-based WAVE2 complex. When these cells were plated on fibronectin-coated dishes, the translocation of WAVE2 to the plasma membrane was significantly reduced and the formation of peripheral lamellipodial structures was disturbed, suggesting that the NESH/Abi-3-based WAVE2 complex was unable to help produce lamellipodial protrusions. Next, Abi-1, Abi-2, or NESH/Abi-3 was expressed in v-src-transformed NIH3T3 cells. Only in NESH/Abi-3-expressed cells did treatment with an Abl kinase inhibitor, imatinib mesylate, or siRNA-mediated knockdown of c-Abl promote the formation of invadopodia, which are ventral membrane protrusions with extracellular matrix degradation activity. Structural studies showed that a linker region between the proline-rich regions and the Src homology 3 (SH3) domain of Abi-1 is crucial for its interaction with c-Abl and c-Abl-mediated phosphorylation of WAVE2. CONCLUSIONS: The NESH/Abi-3-based WAVE2 complex is functionally distinct from the Abi-1-based one, and NESH/Abi-3 may be involved in the formation of ventral protrusions under certain conditions.

γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways.

A role for the ancient SNARE syntaxin 17 in regulating mitochondrial division.

Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.

Late-onset spastic ataxia phenotype in a patient with a homozygous DDHD2 mutation.

Autosomal recessive cerebellar ataxias and autosomal recessive hereditary spastic paraplegias (ARHSPs) are clinically and genetically heterogeneous neurological disorders. Herein we describe Japanese siblings with a midlife-onset, slowly progressive type of cerebellar ataxia and spastic paraplegia, without intellectual disability. Using whole exome sequencing, we identified a homozygous missense mutation in DDHD2, whose mutations were recently identified as the cause of early-onset ARHSP with intellectual disability. Brain MRI of the patient showed a thin corpus callosum. Cerebral proton magnetic resonance spectroscopy revealed an abnormal lipid peak in the basal ganglia, which has been reported as the hallmark of DDHD2-related ARHSP (SPG 54). The mutation caused a marked reduction of phospholipase A1 activity, supporting that this mutation is the cause of SPG54. Our cases indicate that the possibility of SPG54 should also be considered when patients show a combination of adult-onset spastic ataxia and a thin corpus callosum. Magnetic resonance spectroscopy may be helpful in the differential diagnosis of patients with spastic ataxia phenotype.

Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics.

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1 is the first identified intracellular phospholipase A1 and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA1 regulates mitochondrial dynamics. PA-PLA1, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA1 on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA1 in testis, PA-PLA1 knock-out mice have a defect in sperm formation. In PA-PLA1-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA1 in the organization of mitochondria during spermiogenesis.

A lysophospholipid acyltransferase antagonist, CI-976, creates novel membrane tubules marked by intracellular phospholipase A1 KIAA0725p.

CI-976 is a lysophospholipid acyltransferase antagonist that is known to affect secretory and endocytic membrane-trafficking pathways likely by increasing the lysophospholipid content in membranes. Our previous study suggested that lysophospholipids formed through the action of an intracellular phospholipase A(1), KIAA0725p (also known as DDHD2 and iPLA(1)γ), may be important for the association of this enzyme with membranes. In this study, we examined the effect of CI-976 on the membrane association of KIAA0725p. While in HeLa cells KIAA0725p is localized in the Golgi and cytosol, in mouse embryonic fibroblasts (MEFs), it was found to be principally localized in the cytosol with some on post-endoplasmic reticulum compartments including the cis-Golgi. Treatment of MEFs with CI-976 induced the redistribution of KIAA0725p to membrane tubules, which were in vicinity to fragmented mitochondria. These tubules were not decorated with canonical organelle markers including Golgi proteins. A human KIAA0725p mutant, which exhibits decreased membrane-binding ability, was also redistributed to membrane structures upon CI-976 treatment. Our data suggest that the association of KIAA0725p with membranes is regulated by lipid metabolism, and that CI-976 may create unique membrane structures that can be marked by KIAA0725p.

Roles of SAM and DDHD domains in mammalian intracellular phospholipase A1 KIAA0725p.

Members of the intracellular phospholipase A1 family of proteins have been implicated in organelle biogenesis and membrane trafficking. The mammalian family comprises three members: phosphatidic acid-preferring phospholipase A1 (PA-PIA1)/DDHD1, p125/Sec23ip and KIAA0725p/DDHD2, all of which have a DDHD domain. PA-PLAI is mostly cytosolic, while KIAA0725p and p125 are more stably associated with the Golgi/endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) and ER exit sites, respectively. Here we show that KIAAO725p and p125 are novel phosphoinositide-binding proteins. Deletion and mutational analyses of KIAAO725p suggested that a sterile alpha-motif (SAM), which is also present inp125, but not in cytosolic PA-PLAI, and the following DDHD domain comprise a minimal region for phosphatidylinositol 4-phosphate (Pl(4)P)-binding. A construct with mutations in the positively charged cluster of the SAM domain is defective in both phosphoinositide-binding and Golgi/ERGIC targeting. Consistent with the view that the Pl(4)P-binding is important for the membrane association of KIAA0725p, expression of phosphoinositide phosphatase Sacd reduces the association of expressed KIAAO725p with membranes. In addition, we show that deletion of the DDHD domain or introduction of point mutations at the conserved aspartate or histidine residues in the domain abolishes the phospholipase activity of KIAAO725p and PA-PLA1. Together, our results suggest that KIAAO725p is targeted to specific organelle membranes in a phosphoinositide-dependent manner, and that its SAM and DDHD domains are essential for its phosphoinositide-binding and phospholipase activity.

The intracellular phospholipase A1 protein family.

Abstract Phospholipase A1 is an enzyme that hydrolyzes phospholipids, producing 2-acyl-lysophospholipids and fatty acids. The intracellular phospholipase A1 (iPLA1) protein family is a relatively recently discovered lipid-metabolizing enzyme family. Lower eukaryotes, such as yeasts and nematodes, and plants have only one iPLA1 protein, whereas mammals have three iPLA1 family proteins (PA-PLA1/DDHD1/iPLA1α, p125/Sec23IP/iPLA1ß and KIAA0725p/DDHD2/iPLA1γ). Mammalian iPLA1 proteins are localized in different cellular compartments, and two of them, p125 and KIAA0725p, have been implicated in membrane trafficking events. Recent gene targeting studies on several organisms showed that iPLA1 family proteins are involved in various physiological functions, including plant shoot gravitropism, epithelial stem cell differentiation and spermiogenesis. In this review, we describe the features of iPLA1 family proteins and recent progress regarding our understanding of their physiological functions.

Sec16B is involved in the endoplasmic reticulum export of the peroxisomal membrane biogenesis factor peroxin 16 (Pex16) in mammalian cells.

Sec16 plays a key role in the formation of coat protein II vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Mammals have two Sec16 isoforms: Sec16A, which is a longer primary ortholog of yeast Sec16, and Sec16B, which is a shorter distant ortholog. Previous studies have shown that Sec16B, as well as Sec16A, defines ER exit sites, where coat protein II vesicles are formed in mammalian cells. Here, we reveal an unexpected role of Sec16B in the biogenesis of mammalian peroxisomes. When overexpressed, Sec16B was targeted to the entire ER, whereas Sec16A was mostly cytosolic. Concomitant with the overexpression of Sec16B, peroxisomal membrane biogenesis factors peroxin 3 (Pex3) and Pex16 were redistributed from peroxisomes to Sec16B-positive ER membranes. Knockdown of Sec16B but not Sec16A by RNAi affected the morphology of peroxisomes, inhibited the transport of Pex16 from the ER to peroxisomes, and suppressed expression of Pex3. These phenotypes were significantly reversed by the expression of RNAi-resistant Sec16B. Together, our results support the view that peroxisomes are formed, at least partly, from the ER and identify a factor responsible for this process.

p125/Sec23-interacting protein (Sec23ip) is required for spermiogenesis.

p125/Sec23ip is a phospholipase A(1)-like protein that interacts with Sec23, a coat component of COPII vesicles that bud from endoplasmic reticulum exit sites. To understand its physiological function, we produced p125 knockout mice. The p125 knockout mice grew normally, but males were subfertile. Sperm from p125-deficient mice had round heads and lacked the acrosome, an organelle containing the enzymes responsible for fertilization. p125 was found to be expressed at stages I-XII of spermatogenesis, similar to the expression pattern of proteins involved in acrosome biogenesis. These results suggest that p125 plays an important role in spermiogenesis.

Dual function of Sec16B: Endoplasmic reticulum-derived protein secretion and peroxisome biogenesis in mammalian cells.

The origin of peroxisomes has long been disputed. However, recent evidence suggests that peroxisomes can be formed de novo from the endoplasmic reticulum (ER) in yeast and higher eukaryotes. Sec16A and Sec16B, mammalian orthologs of yeast Sec16, are scaffold proteins that organize ER exit sites by interacting with COPII components. We recently demonstrated that Sec16B, but not Sec16A, regulates the transport of peroxisomal biogenesis factors from the ER to peroxisomes in mammalian cells. The C-terminal region of Sec16B, which is not conserved in Sec16A, is required for this function. The data suggest that Sec16B in ER areas other than ER exit sites plays this role. Our findings provide an unexpected connection between at least part of the COPII machinery and the formation of preperoxisomal vesicles at the ER, and offer an explanation of how secretory and peroxisomal trafficking from the ER are distinguished.

Golgi-localized KIAA0725p regulates membrane trafficking from the Golgi apparatus to the plasma membrane in mammalian cells.

Mammals have three members of the intracellular phospholipase A(1) protein family (phosphatidic acid preferring-phospholipase A(1), p125, and KIAA0725p). In this study, we showed that KIAA0725p is localized in the Golgi, and is rapidly cycled between the Golgi and cytosol. Catalytic activity is important for targeting of KIAA0725p to Golgi membranes. RNA interference experiments suggested that KIAA0725p contributes to efficient membrane trafficking from the Golgi apparatus to the plasma membrane, but is not involved in brefeldin A-induced Golgi-to-endoplasmic reticulum retrograde transport.

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains.

The presence of subdomains in the endoplasmic reticulum (ER) enables this organelle to perform a variety of functions, yet the mechanisms underlying their organization are poorly understood. In the present study, we show that syntaxin 18, a SNAP (soluble NSF attachment protein) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. Knockdown of syntaxin 18 caused a global change in ER membrane architecture, leading to the segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed concomitantly with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.

UBXD1 is a VCP-interacting protein that is involved in ER-associated degradation.

AAA ATPase VCP and its yeast ortholog Cdc48, in a complex with the Ufd1-Npl4 heterodimer as an adaptor, play an essential role in endoplasmic reticulum-associated degradation (ERAD). Several UBX domain-containing proteins function to recruit ubiquitylated substrates to VCP/Cdc48 by binding both VCP/Cdc48 and other ERAD components such as ubiquitin ligases. Here we show that mammalian UBXD1 is an additional UBX domain-containing protein involved in the ERAD process. UBXD1 is a cytosolic protein that interacts with VCP and Derlin-1. Overexpression of UBXD1 in cells causes selective dissociation of Ufd1 from VCP, resulting in inhibition of mutant cystic fibrosis transmembrane conductance regulator (CFTR) degradation by ERAD. Additionally, depletion of endogenous UBXD1 protein by RNA interference also results in a defect in CFTR degradation. Collectively, these findings suggest that UBXD1 is a regulatory component of ERAD that may modulate the adaptor binding to VCP.

Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load.

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIalpha (PI4K-IIIalpha) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIalpha, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

Bap31 is an itinerant protein that moves between the peripheral endoplasmic reticulum (ER) and a juxtanuclear compartment related to ER-associated Degradation.

Certain endoplasmic reticulum (ER)-associated degradation (ERAD) substrates with transmembrane domains are segregated from other ER proteins and sorted into a juxtanuclear subcompartment, known as the ER quality control compartment. Bap31 is an ER protein with three transmembrane domains, and it is assumed to be a cargo receptor for ER export of some transmembrane proteins, especially those prone to ERAD. Here, we show that Bap31 is a component of the ER quality control compartment and that it moves between the peripheral ER and a juxtanuclear ER or ER-related compartment distinct from the conventional ER-Golgi intermediate compartment. The third and second transmembrane domains of Bap31 are principally responsible for the movement to and recycling from the juxtanuclear region, respectively. This cycling was blocked by depolymerization of microtubules and disruption of dynein-dynactin function. Overexpression of Sar1p and Arf1 mutants affected Bap31 cycling, suggesting that this cycling pathway is related to the conventional vesicular transport pathways.

Sec22b-dependent assembly of endoplasmic reticulum Q-SNARE proteins.

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved alpha-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in alpha-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.