A PH Domain in the Arf GTPase-activating Protein (GAP) ARAP1 Binds Phosphatidylinositol 3,4,5-Trisphosphate and Regulates Arf GAP Activity Independently of Recruitment to the Plasma Membranes

ARAP1 is a phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GTPase-activating protein (GAP) with five PH domains that regulates endocytic trafficking of the epidermal growth factor receptor (EGFR). Two tandem PH domains are immediately N-terminal of the Arf GAP domain, and one of these fits the consensus sequence for PtdIns(3,4,5)P₃ binding. Here, we tested the hypothesis that PtdIns(3,4,5)P₃-dependent recruitment mediated by the first PH domain of ARAP1 regulates the in vivo and in vitro function of ARAP1. We found that PH1 of ARAP1 specifically bound to PtdIns(3,4,5)P₃, but with relatively low affinity (≈1.6 μm), and the PH domains did not mediate PtdIns(3,4,5)P₃-dependent recruitment to membranes in cells. However, PtdIns(3,4,5)P₃ binding to the PH domain stimulated GAP activity and was required for in vivo function of ARAP1 as a regulator of endocytic trafficking of the EGFR. Based on these results, we propose a variation on the model for the function of phosphoinositide-binding PH domains. In our model, ARAP1 is recruited to membranes independently of PtdIns(3,4,5)P₃, the subsequent production of which triggers enzymatic activity.Pleckstrin homology (PH)² domains are a common structural motif encoded by the human genome (1, 2). Approximately 10% of PH domains bind to phosphoinositides. These PH domains are thought to mediate phosphoinositide-dependent recruitment to membranes (1–3). Most PH domains likely have functions other than or in addition to phosphoinositide binding. For example, PH domains have been found to bind to protein and DNA (4–12). In addition, some PH domains have been found to be structurally and functionally integrated with adjacent domains (13, 14). A small fraction of PH domain-containing proteins (about 9% of the human proteins) have multiple PH domains arranged in tandem, which have been proposed to function as adaptors but have only been examined in one protein (15, 16). Arf GTPase-activating proteins (GAPs) of the ARAP family are phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P₃)-dependent Arf GAPs with tandem PH domains (17, 18). The function of specific PH domains in regulating Arf GAP activity and for biologic activity has not been described.Arf GAPs are proteins that induce the hydrolysis of GTP bound to Arfs (19–23). The Arf proteins are members of the Ras superfamily of GTP-binding proteins (24–27). The six Arf proteins in mammals (five in humans) are divided into three classes based on primary sequence: Arf1, -2, and -3 are class 1, Arf4 and -5 are class 2, and Arf6 is class 3 (23, 24, 27–29). Class 1 and class 3 Arf proteins have been studied more extensively than class 2. They have been found to regulate membrane traffic and the actin cytoskeleton.The Arf GAPs are a family of proteins with diverse domain structures (20, 21, 23, 30). ARAPs, the most structurally complex of the Arf GAPs, contain, in addition to an Arf GAP domain, the sterile α motif (SAM), five PH, Rho GAP, and Ras association domains (17, 18, 31, 32). The first and second and the third and fourth PH domains are tandem (Fig. 1). The first and third PH domains of the ARAPs fit the consensus for PtdIns(3,4,5)P₃ binding (33–35). ARAPs have been found to affect actin and membrane traffic (21, 23). ARAP3 regulates growth factor-induced ruffling of porcine aortic endothelial cells (31, 36, 37). The function is dependent on the Arf GAP and Rho GAP domains. ARAP2 regulates focal adhesions, an actin cytoskeletal structure (17). ARAP2 function requires Arf GAP activity and a Rho GAP domain capable of binding RhoA·GTP. ARAP1 has been found to have a role in membrane traffic (18). The protein associates with pre-early endosomes involved in the attenuation of EGFR signals. The function of the tandem PH domains in the ARAPs has not been examined.Open in a separate windowFIGURE 1.ARAP1 binding to phospholipids. A, schematic of the recombinant proteins used in this study. Domain abbreviations: Ank, ankyrin repeat; PLCδ-PH, PH domain of phospholipase C δ; RA, Ras association motif; RhoGAP, Rho GTPase-activating domain. B, ARAP1 phosphoinositide binding specificity. 500 nm PH1-Ank recombinant protein was incubated with sucrose-loaded LUVs formed by extrusion through a 1-μm pore filter. LUVs contained PtdIns alone or PtdIns with 2.5 μm PtdIns(3,4,5)P₃, 2.5 μm PtdIns(3)P, 2.5 μm PtdIns(4)P, 2.5 μm PtdIns(5)P, 2.5 μm PtdIns(3,4)P₂, 2.5 μm PtdIns(3,5)P₂, or 2.5 μm PtdIns(4,5)P₂ with a total phosphoinositide concentration of 50 μm and a total phospholipid concentration of 500 μm. Vesicles were precipitated by ultracentrifugation, and associated proteins were separated by SDS-PAGE. The amount of precipitated protein was determined by densitometry of the Coomassie Blue-stained gels with standards on each gel. C, PtdIns(3,4,5)P₃-dependent binding of ARAP1 to LUVs. 1 μm PH1-Ank or ArfGAP-Ank recombinant protein was incubated with 1 mm sucrose-loaded LUVs formed by extrusion through a 1-μm pore size filter containing varying concentration of PtdIns(3,4,5)P₃. Precipitation of LUVs and analysis of associated proteins were performed as described in B. The average ± S.E. of three independent experiments is presented.Here we investigated the role of the first two PH domains of ARAP1 for catalysis and in vivo function. The first PH domain fits the consensus sequence for PtdIns(3,4,5)P₃ binding (33–35). The second does not fit a phosphoinositide binding consensus but is immediately N-terminal to the GAP domain. We have previously reported that the PH domain that occurs immediately N-terminal of the Arf GAP domain of ASAP1 is critical for the catalytic function of the protein (38, 39). We tested the hypothesis that the two PH domains of ARAP1 function independently; one recruits ARAP1 to PtdIns(3,4,5)P₃-rich membranes, and the other functions with the catalytic domain. As predicted, PH1 interacted specifically with PtdIns(3,4,5)P₃, and PH2 did not. However, both PH domains contributed to catalysis independently of recruitment to membranes. None of the PH domains in ARAP1 mediated PtdIns(3,4,5)P₃-dependent targeting to plasma membranes (PM). PtdIns(3,4,5)P₃ stimulated GAP activity, and the ability to bind PtdIns(3,4,5)P₃ was required for ARAP1 to regulate membrane traffic. We propose that ARAP1 is recruited independently of PtdIns(3,4,5)P₃ to the PM where PtdIns(3,4,5)P₃ subsequently regulates its GAP activity to control endocytic events.     

The Clavesin Family, Neuron-specific Lipid- and Clathrin-binding Sec14 Proteins Regulating Lysosomal Morphology

Clathrin-coated vesicles (CCVs) originating from the trans-Golgi network (TGN) provide a major transport pathway from the secretory system to endosomes/lysosomes. Herein we describe paralogous Sec14 domain-bearing proteins, clavesin 1/CRALBPL and clavesin 2, identified through a proteomic analysis of CCVs. Clavesins are enriched on CCVs and form a complex with clathrin heavy chain (CHC) and adaptor protein-1, major coat components of TGN-derived CCVs. The proteins co-localize with markers of endosomes and the TGN as well as with CHC and adaptor protein-1. A membrane mimic assay using the Sec14 domain of clavesin 1 reveals phosphatidylinositol 3,5-bisphosphate as a specific lipid partner. Phosphatidylinositol 3,5-bisphosphate is localized to late endosomes/lysosomes, and interestingly, isoform-specific knockdown of clavesins in neurons using lentiviral delivery of interfering RNA leads to enlargement of a lysosome-associated membrane protein 1-positive membrane compartment with no obvious influence on the CCV machinery at the TGN. Since clavesins are expressed exclusively in neurons, this new protein family appears to provide a unique neuron-specific regulation of late endosome/lysosome morphology.Proteins entering the secretory pathway move through the Golgi apparatus to the trans-Golgi network (TGN)⁴ where they are sorted and packaged into carrier vesicles, including clathrin-coated vesicles (CCVs) for transport to their final destination (1). Adaptor protein-1 (AP-1), which is recruited to the TGN through dual interactions with Arf1 and phosphatidylinositol 4-phosphate (2), recruits clathrin to initiate CCV formation. AP-1 and clathrin form the membrane coat that shapes the vesicle and recruits an array of regulatory/accessory proteins, which control numerous aspects of CCV formation and function (3). Interactions with the clathrin·AP-1 coat complex also serve to recruit both transmembrane and cytosolic cargo to CCVs, allowing for their transport from the TGN to the endosomal network.The specificity and function of intracellular compartments depends in part on the presence of distinct PtdIns species. For example, phosphatidylinositol 4-phosphate is found predominantly at the TGN and contributes to AP-1 recruitment, whereas phosphatidylinositol 3-phosphate recruits effectors, such as EEA1 (early endosome antigen 1), to early endosomes to mediate membrane fusion (4–9). Early endosomes subsequently transition into late endosomes/lysosomes, and during this process, phosphatidylinositol 3-phosphate is converted to PtdIns(3,5)P₂ via the action of a phosphatidylinositol 3-phosphate 5-kinase named Fab1p in yeast and PIKfyve in mammals (10). PIKfyve is part of a protein complex nucleated by Vac14 (11, 12), and disruption of this complex leads to decreased PtdIns(3,5)P₂ levels and the formation of enlarged cytoplasmic vacuoles of endosomal/lysosomal origin (10, 13, 14). Intriguingly, despite the fact that Vac14 is found in all tissues (15) and regulates a ubiquitous trafficking process (10), decreases in PtdIns(3,5)P₂ levels resulting from Vac14 knock-out show massive neurodegeneration with little effect on other tissues (16). This neuron-specific effect has remained mysterious due to the lack of exclusively neuronal factors targeting PtdIns(3,5)P₂.The Sec14 domain is an evolutionarily ancient protein module that in humans is found in more than 45 proteins encoded by at least 25 genes (17). Mutations in several Sec14 proteins lead to human diseases, including neurodegeneration (18), yet most of the Sec14 proteins in mammals remain uncharacterized. The yeast protein Sec14p is the prototype for this module (19). Sec14p is essential for the transport of proteins from the Golgi (19), but more recently it was also found to be involved in the trafficking of protein cargo, specifically the mating factor receptor Ste3, from the plasma membrane via endosomes to the yeast vacuole (17). The yeast vacuole is the equivalent of the lysosome in mammalian cells. Yeast Sec14p is a phospholipid transfer protein that extracts phosphatidylinositol and phosphatidylcholine from membranes in vitro and regulates the metabolism of these lipids in cells (19, 20). Sec14p is composed of the Sec14 domain only, whereas the majority of Sec14 proteins in mammals contain a Sec14 domain in conjunction with other modules or protein/lipid binding domains (21, 22). In the few well characterized examples, these proteins function to integrate lipid binding/metabolism with other biological functions (21, 22).In a subcellular proteomic analysis of CCVs from the brain, we identified eight novel open reading frames, including one encoding an Sec14 domain (23, 24). A subsequent bioinformatics analysis revealed a second, paralogous Sec14 protein. We now demonstrate that the proteins, which we have named clavesin (clathrin vesicle-associated Sec14 protein) 1 and 2, are neuron-specific proteins that function in the regulation of lysosome morphology.     

The Vac14-interaction Network Is Linked to Regulators of the Endolysosomal and Autophagic Pathway

The scaffold protein Vac14 acts in a complex with the lipid kinase PIKfyve and its counteracting phosphatase FIG4, regulating the interconversion of phosphatidylinositol-3-phosphate to phosphatidylinositol-3,5-bisphosphate. Dysfunctional Vac14 mutants, a deficiency of one of the Vac14 complex components, or inhibition of PIKfyve enzymatic activity results in the formation of large vacuoles in cells. How these vacuoles are generated and which processes are involved are only poorly understood. Here we show that ectopic overexpression of wild-type Vac14 as well as of the PIKfyve-binding deficient Vac14 L156R mutant causes vacuoles. Vac14-dependent vacuoles and PIKfyve inhibitor-dependent vacuoles resulted in elevated levels of late endosomal, lysosomal, and autophagy-associated proteins. However, only late endosomal marker proteins were bound to the membranes of these enlarged vacuoles. In order to decipher the linkage between the Vac14 complex and regulators of the endolysosomal pathway, a protein affinity approach combined with multidimensional protein identification technology was conducted, and novel molecular links were unraveled. We found and verified the interaction of Rab9 and the Rab7 GAP TBC1D15 with Vac14. The identified Rab-related interaction partners support the theory that the regulation of vesicular transport processes and phosphatidylinositol-modifying enzymes are tightly interconnected.Lipid kinases and phosphatases tightly regulate the interconversion and abundance of different phosphoinositide lipid derivatives (PIPs),¹ which are crucial components for the identity of eukaryotic membranes (1, 2). PIPs and their modifying proteins control multiple cellular functions such as signal transduction, cytoskeletal dynamics, and membrane trafficking (1, 2). Synthesis and turnover of the low-abundant lipid phosphatidylinositol-3,5-bisphosphate is controlled by the Vac14 complex. This complex contains the scaffold protein Vac14 (ArPIKfyve), the lipid kinase PIKfyve (Fab1), and its counteracting lipid phosphatase FIG4 (SAC3) (3, 4).The overall protein structure of Vac14 is conserved from yeast to human and consists of multiple tandem HEAT (huntingtin, elongation Factor 3, PR65/A, TOR) repeats and a rod-like helical domain that controls protein–protein interactions (5, 6). Previous studies showed that the aminoterminal part of Vac14 mediates the binding to PIKfyve. The carboxyterminal region is crucial for the interaction with FIG4 and also contains a PDZ-binding motif, which binds to nNOS (5, 7–11). Moreover, Vac14-oligomerization, which is essential for the nucleation of an active Vac14-complex, overlaps with the FIG4 interaction binding site (5, 6, 12, 13).The lack of Vac14 or the ectopic overexpression of a PIKfyve binding-deficient Vac14 mutant in vivo results in a reduced level of phosphatidylinositol-3,5-bisphosphate and is accompanied by enhanced formation of enlarged intracellular vesicles, hereinafter called vacuoles (5, 14, 15). This was initially observed in yeast, where it leads to enlargement of the yeast vacuole, which is comparable to the mammalian lysosome (12, 16, 17). The vacuoles in mammalian cells are heterogeneous, positive for early or late endosomal structures, and involved in vesicular trafficking processes from the late endosome to the trans-Golgi network (14, 18, 19).We now report that in addition to overexpression of the PIKfyve binding deficient mutant, overexpression of the Vac14 wild type was sufficient to induce vacuolization in human cell cultures. The Vac14-based vacuolization leads to a significant accumulation of predominantly late endosomal and autophagosomal marker proteins with only late endosomal proteins decorating the vacuolar membranes. These results almost completely phenocopy previously described effects of the inhibitor YM201636, which specifically blocks PIKfyve lipid kinase activity (20–24).To identify proteins involved in Vac14-induced vacuolization, we used a protein affinity approach combined with multidimensional protein identification technology (MudPIT). The evaluation of the identified peptides elucidated numerous potential Vac14 interacting proteins involved in intracellular trafficking and membrane dynamics, with Rab9 and TBC1D15 being the most promising candidates. The specificity of the Vac14 interaction with Rab9 and the Rab7 regulator TBC1D15 was confirmed by co-immunoprecipitation assays. Moreover, we were able to demonstrate that Rab9 accumulates during vacuolization and localizes on the limiting membranes of vacuoles as a result of Vac14 overexpression. In summary, the identification of Rab9 and TBC1D15 as novel interaction partners provides new insights into the molecular functions of Vac14 in vesicular transport processes.     

Profiling Lipid–protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Proteome Microarrays

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)¹ liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 μm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P₂-specific binding proteins (PI(3,5)P₂-BPs). We not only recovered many proteins that possessed known PI(3,5)P₂-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P₂-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P₂-BPs were similar to those associated with PI(3,5)P₂, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P₂-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P₂ interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P₂-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid–protein interactions in high-throughput studies.Cell viability and physiological functions are maintained through a complex biomolecular interaction network. One of the key components in the regulatory system includes lipid–protein interactions that mediate various cell responses and metabolisms. Increasing evidence shows that such interactions have profound influences on cell polarization, the cell cycle, and other cellular processes. To date, in vitro characterizations of lipid interactions with other biomolecules are often conducted using artificial membrane models, such as liposomal nanovesicles, to mimic biological membranes. Liposomal nanovesicles, termed liposomes, are spherical vesicles that are surrounded by phospholipid bilayers in which the lipid of interest can be incorporated. An important benefit of liposomes is the ease in which a large number of fluorescent molecules can be encapsulated so that the liposome binding signals can be greatly enhanced for detection (1–4). Therefore, liposomes have become a practical and popular tool for use as a model membrane or fluorophore-loaded vehicle to study signal amplification (1–4) and/or lipid research (5–9).In general, liposomes are capable of encapsulating hundreds of millions of fluorescent dye molecules, thereby providing greatly enhanced signals (1–4). However, high concentrations of fluorophores often lead to self-quenching, and as a result, the fluorescent signals cannot be detected without first lysing the liposomes (1–4). This issue has limited their applications for real-time detection and high-density chip assays. To solve this problem, we developed a novel non-quenched fluorescent (NQF) liposome with the capability of signal amplification. During the fabrication procedure, we used sulforhodamine B (SRB) as an encapsulant and incorporated lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) within the liposomal bilayer.Profiling phosphatidylinositide-protein interactions is of particular interest because these lipids have been implicated in a wide variety of cell functions, including cell signaling, actin cytoskeletal reorganization, exocytosis, and intracellular trafficking (10–14). Among the phosphatidylinositides, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂) is one of the most important mediators of signal transduction (15, 16). Intensive studies over the past decade have shown that PI(3,5)P₂ is involved in protein sorting into multivesicular bodies (MVBs), membrane recycling/turnover, and the vacuole acidification (17–19). Like other phosphatidylinositides, PI(3,5)P₂ may regulate downstream pathways through the binding of the myo-inositol head group to proteins containing phosphoinositide-binding domains (20, 21). Thus far, a handful of modular phosphoinositide-binding domains have been identified, including C2 (Protein Kinase C homology 2) (22), a WD-40 motif (tryptophan-aspartic acid repeats) that folds as β-propellers (23), ARRB1 (β-arrestin 1), and a number of actin regulatory domains (e.g. the gelsolin/villin family, cofilin, and profilin) (20).To globally profile PI(3,5)P₂-binding proteins as the foundation for a better understanding of the biology of PI(3,5)P₂, we employed the newly developed PI(3,5)P₂-NQF liposomes to probe the Saccharomyces cerevisiae proteome microarray. We not only recovered many proteins that contained known PI(3,5)P₂-binding domains, but we also validated many newly discovered PI(3,5)P₂-binding proteins using a bead-based affinity assay. Representing both a signal and an analyte carrier, the NQF liposomes should provide a new research model for studying lipid–protein interactions in the future.     

Regulation of Phosphatidylinositol Kinases and Metabolism by Wnt3a and Dvl

Wnt signaling plays important roles in various physiological and pathophysiological processes. The pathway that leads to β-catenin stabilization is initiated by Wnt binding to its cell surface receptors, which induces the formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) via activation of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) type I. Here, we show that Wnt also stimulated the production of phosphatidylinositol 4-phosphate (PtdIns(4)P), which depended on Frizzled (Fz), Dishevelled (Dvl), and phosphatidylinositol 4-kinase (PI4K) type IIα in HEK293T cells. Dvl directly interacted with and activated PI4KIIα by increasing its V_max for ATP and PtdIns. In addition, Dvl regulated PI4KIIα and PIP5KI via different domains. Moreover, Dvl, PI4KIIα, and PIP5KI appeared to form a ternary complex upon Wnt3a stimulation. This complex may allow efficient production of PtdIns(4,5)P₂ from PtdIns, which is far more abundant than PtdIns(4)P in cells. Therefore, this study provides new insights into the mechanism by which Wnt3a regulates the production of PtdIns(4,5)P₂.The Wnt family of secretory glycoproteins plays important roles in regulation of embryonic development and tumorigenesis. They also regulate many other physiological and pathophysiological processes, including bone development, neuronogenesis, adipogenesis, myogenesis, organogenesis, and lipid and glucose metabolism (1–5). Studies using Drosophila and Xenopus embryos as well as mammalian cells have established a canonical Wnt signaling pathway that leads to stabilization of β-catenin. In the absence of Wnt, a number of proteins, including Axin, adenomatous polyposis coli (APC), casein kinase 1 (CK1), glycogen synthase kinase-3β (GSK3β),³ form a complex that facilitates β-catenin phosphorylation by CK1 and GSK3β. This phosphorylation targets β-catenin for ubiquitination and proteasome-mediated proteolytic degradation (3, 6). Some of the Wnt proteins bind to two cell surface receptors Fz and low density lipoprotein receptor-related protein (LRP) 5/6 and initiate a signaling cascade that eventually leads to the suppression of β-catenin phosphorylation by GSK3β and stabilization of β-catenin.Because the finding that the canonical Wnt proteins transduce signals by inducing the interaction between LRP5/6 and Axin (7), more has been learned about the mechanisms by which this interaction is regulated by Wnt proteins. Studies have indicated that two phosphorylation events at the C-terminal intracellular domain of LRP5/6, the phosphorylation of Thr¹⁴⁷⁹ by CKIγ (8, 9) and of Ser¹⁴⁹⁰ by GSK3 (10, 11), were required for the interaction. We recently showed that Wnt3a stimulated the production of PtdIns (4,5)P₂, which in turn regulated the phosphorylation of LRP5/6 at Thr¹⁴⁷⁹ and Ser¹⁴⁹⁰ (12). We also showed that Wnt3a regulated phosphatidylinositol 4-phosphate 5-kinase type I (PIP5KI) activity by inducing the interaction between Dvl and PIP5KI (12). Moreover, Dvl could directly stimulate the lipid kinase activity of PIP5KI (12).PtdIns(4,5)P₂ plays important roles in various cellular functions, including membrane trafficking, cytoskeletal reorganization, migration, ion channel activation, and signal transduction (13). It, however, represents less than 1% of plasma membrane phospholipids and is primarily synthesized in most cells by sequential phosphorylation of PtdIns on the D4 and D5 positions of the inositol ring by two PtdIns kinases, PI4K and PIP5KI, respectively (14, 15). While PtdIns(4)P, the substrate for PIP5KI, is also accounted for around 1% of plasma membrane phospholipids, PtdIns, the substrate for PI4K, is very abundant. Thus, Wnt3a may have to stimulate PI4K activity to provide enough substrate for PIP5KI in PtdIns(4,5)P₂ production.Two types of PI4K (PI4KI and PI4KII) have been characterized in mammalian cells. There are two isoforms of PI4KII (PI4KIIα and PI4KIIβ) and two isoforms of PI4KI (PI4KIα and PI4KIβ) (16). In our previous study, we demonstrated the involvement of PI4KIIα in Wnt signaling. siRNA-mediated knockdown in mammalian cells and morpholino-mediated suppression in Xenopus embryos of PI4KIIα inhibited LRP6 phosphorylation and Wnt signaling. In this report, we examined whether Wnt3a regulates the lipid kinase activity of PI4KIIα and found that Wnt3a could induce an increase in the level of PtdIns(4)P in a Dvl- and Fz-dependent manner. In addition, the Dvl protein was found to directly interact with and activate PI4KIIα. Moreover, different domains of Dvl appeared to be involved in the regulation of PI4KIIα and PIP5KI, and Wnt3a induced the formation of a complex of Dvl, PI4KIIα, and PIP5KI possibly for more efficient production of PtdIns (4,5)P₂ in cells.     

Localized Diacylglycerol-dependent Stimulation of Ras and Rap1 during Phagocytosis

We describe a role for diacylglycerol in the activation of Ras and Rap1 at the phagosomal membrane. During phagocytosis, Ras density was similar on the surface and invaginating areas of the membrane, but activation was detectable only in the latter and in sealed phagosomes. Ras activation was associated with the recruitment of RasGRP3, a diacylglycerol-dependent Ras/Rap1 exchange factor. Recruitment to phagosomes of RasGRP3, which contains a C1 domain, parallels and appears to be due to the formation of diacylglycerol. Accordingly, Ras and Rap1 activation was precluded by antagonists of phospholipase C and of diacylglycerol binding. Ras is dispensable for phagocytosis but controls activation of extracellular signal-regulated kinase, which is partially impeded by diacylglycerol inhibitors. By contrast, cross-activation of complement receptors by stimulation of Fcγ receptors requires Rap1 and involves diacylglycerol. We suggest a role for diacylglycerol-dependent exchange factors in the activation of Ras and Rap1, which govern distinct processes induced by Fcγ receptor-mediated phagocytosis to enhance the innate immune response.Receptors that interact with the constant region of IgG (FcγR)⁴ mediate the recognition and elimination of soluble immune complexes and particles coated (opsonized) with immunoglobulins. Clustering of FcγR on the surface of leukocytes upon attachment to multivalent ligands induces their activation and subsequent internalization. Soluble immune complexes are internalized by endocytosis, a clathrin- and ubiquitylation-dependent process (1). In contrast, large, particulate complexes like IgG-coated pathogens are ingested by phagocytosis, a process that is contingent on extensive actin polymerization that drives the extension of pseudopods (2). In parallel with the internalization of the opsonized targets, cross-linking of phagocytic receptors triggers a variety of other responses that are essential components of the innate immune response. These include degranulation, activation of the respiratory burst, and the synthesis and release of multiple inflammatory agents (3, 4).Like T and B cell receptors, FcγR possesses an immunoreceptor tyrosine-based activation motif that is critical for signal transduction (3, 4). Upon receptor clustering, tyrosyl residues of the immunoreceptor tyrosine-based activation motif are phosphorylated by Src family kinases, thereby generating a docking site for Syk, a tyrosine kinase of the ZAP70 family (3, 4). The recruitment and activation of Syk in turn initiates a cascade of events that include activation of Tec family kinases, Rho- and ARF-family GTPases, phosphatidylinositol 3-kinase, phospholipase Cγ (PLCγ), and a multitude of additional effectors that together remodel the underlying cytoskeleton, culminating in internalization of the bound particle (5, 6).Phosphoinositide metabolism is thought to be critical for FcγR-induced phagocytosis (7, 8). Highly localized and very dynamic phosphoinositide changes have been observed at sites of phagocytosis: phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) undergoes a transient accumulation at the phagocytic cup, which is rapidly superseded by its complete elimination from the nascent phagosome (7). The secondary disappearance of PtdIns(4,5)P₂ is attributable in part to the localized generation of phosphatidylinositol 3,4,5-trisphosphate, which has been reported to accumulate at sites of phagocytosis (9). Activation of PLCγ is also believed to contribute to the acute disappearance of PtdIns(4,5)P₂ in nascent phagosomes. Indeed, the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate has been detected by chemical means during FcγR-evoked particle ingestion (10, 11). Moreover, imaging experiments revealed that DAG appears at the time and at the precise site where PtdIns(4,5)P₂ is consumed (7).Two lines of evidence suggest that the DAG generated upon engagement of phagocytic receptors modulates particle engulfment. First, antagonists of PLC severely impair phagocytosis by macrophages (7, 12). This inhibition is not mimicked by preventing the associated [Ca²⁺] transient, suggesting that DAG, and not inositol 1,4,5-trisphosphate, is the crucial product of the PLC (13). Second, the addition of exogenous DAG or phorbol esters, which mimic the actions of endogenous DAG, augment phagocytosis (14, 15).Selective recognition of DAG by cellular ligands is generally mediated by specific regions of its target proteins, called C1 domains (16). Proteins bearing C1 domains include, most notably, members of the classical and novel families of protein kinase C (PKC), making them suitable candidates to account for the DAG dependence of phagocytosis. Indeed, PKCα, a classical isoform, and PKCϵ and PKCδ, both novel isoforms, are recruited to phagosomes (12, 15, 17, 18). Although the role of the various PKC isoforms in particle engulfment has been equivocal over the years, Cheeseman et al. (12) convincingly demonstrated that PKCϵ contributes to particle uptake in a PLC- and DAG-dependent manner.PKCs are not the sole proteins bearing DAG-binding C1 domains. Similar domains are also found in several other proteins, including members of the RasGRP family, chimaerins, and Munc-13 (19–21). One or more of these could contribute to the complex set of responses elicited by FcγR-induced DAG production. The RasGRP proteins are a class of exchange factors for the Ras/Rap family of GTPases (22). There are four RasGRP proteins (RasGRP1 to -4), and emerging evidence has implicated RasGRP1 and RasGRP3 in T and B cell receptor signaling (23–27).The possible role of DAG-mediated signaling pathways other than PKC in phagocytosis and the subsequent inflammatory response has not been explored. Here, we provide evidence that DAG stimulates Ras and Rap1 at sites of phagocytosis, probably through RasGRPs. Last, the functional consequences of Ras and Rap1 activation were analyzed.     

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca²⁺ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca²⁺ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca²⁺ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P₂ depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca²⁺ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced I_CRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P₂ is a likely determinant of Orai1 channel activity.Store-operated Ca²⁺ entry (SOCE)³ is a ubiquitous Ca²⁺ entry pathway that is regulated by the Ca²⁺ content of the endoplasmic reticulum (ER) (1). SOCE has been identified as the major route of Ca²⁺ entry during activation of cells of the immune system such as T cells and mast cells (2, 3), and it is also present and functionally important in other cells such as platelets (4) and developing myotubes (5). The long awaited mechanism of how the ER luminal Ca²⁺ content is sensed and the information transferred to the plasma membrane (PM) has been clarified recently after identification of the ER Ca²⁺ sensor proteins STIM1 and -2 (6, 7) and the PM Ca²⁺ channels Orai1, -2, and -3 (8–10). According to current views, a decrease in the ER Ca²⁺ concentration is sensed by the luminal EF-hand of the single-transmembrane STIM proteins causing their multimerization. This oligomerization occurs in the tubular ER, where it promotes the interaction of the cytoplasmic C termini of STIM with PM components and association with the PM-localized Orai channels, causing both their clustering and activation in the PM (reviewed recently in Refs. 11–13). Analysis of the interacting domains within the STIM1 and Orai1 proteins suggests that the cytoplasmic domain of STIM1 is necessary and sufficient to activate Orai1 (14), whereas the latter requires its C-terminal membrane-adjacent cytoplasmic tail to be fully activated by the STIM proteins (15, 16). Both STIM1 and -2 contain a polybasic segment in their C termini, and such regions are often responsible for the PM localization of proteins (mostly of the small GTP-binding protein class) via interaction with anionic phospholipids such as phosphatidylserine or PtdIns(4,5)P₂ (17). However, the role of this domain in STIM1 function(s) remains controversial. Deletion of the polybasic tail is reported to prevent PM association but not clustering of STIM1 upon ER store depletion (18). In other studies, truncated STIM1 lacking the polybasic domain shows only slightly altered activation (15) or inactivation (19) kinetics without major defects in supporting Orai1-mediated Ca²⁺ influx. The most recent studies identify the minimal Orai1 activation domain in STIM1 (20, 21) and find that the polybasic domain is not essential for this function but makes electrostatic interaction with classical transient receptor potential channels (22).PM phosphoinositides have been widely reported as regulators of the activity of several ion channels and transporters (23). However, only a few studies have addressed the inositide requirement of SOCE and none specifically that of the Orai1-mediated Ca²⁺ entry process. Sensitivity of SOCE to phosphatidylinositol 3-kinases (PI3K) inhibitors has been reported, but this required concentrations that suggested inhibition of targets other than PI3Ks, possibly myosin light chain kinase or the type-III PI4Ks (4, 24–26). Here we have described studies addressing the role of PM phosphoinositides in STIM1 movements as well as in Orai1 channel gating. Our results show that phosphoinositides do not have a major role in the prominent reorganization of STIM1 after Ca²⁺ store depletion but suggest a function of PtdIns4P rather than PtdIns(4,5)P₂ in supporting the Orai1-mediated Ca²⁺ entry process.     

Kinetics of the Interaction of myo1c with Phosphoinositides

myo1c is a single-headed myosin that dynamically links membranes to the actin cytoskeleton. A putative pleckstrin homology domain has been identified in the myo1c tail that binds phosphoinositides and soluble inositol phosphates with high affinity. However, the kinetics of association and dissociation and the influence of phospholipid composition on the kinetics have not been determined. Stopped-flow spectroscopy was used to measure the binding and dissociation of a recombinant myo1c construct containing the tail and regulatory domains (myo1c^IQ-tail) to and from 100-nm diameter large unilamellar vesicles (LUVs). We found the time course of association of myo1c^IQ-tail with LUVs containing 2% phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) followed a two-exponential time course, and the rate of the predominant fast phase depended linearly upon the total lipid concentration. The apparent second-order rate constant was approximately diffusion-limited. Increasing the molar ratio of anionic phospholipid by adding phosphatidylserine, additional PtdIns(4,5)P₂, or by situating PtdIns(4,5)P₂ in a more physiologically relevant lipid background increased the apparent association rate constant less than 2-fold. myo1c^IQ-tail dissociated from PtdIns(4,5)P₂ at a slower rate (2.0 s⁻¹) than the pleckstrin homology domain of phospholipase C-δ (13 s⁻¹). The presence of additional anionic phospholipid reduced the myo1c^IQ-tail dissociation rate constant >50-fold but marginally changed the dissociation rate of phospholipase C-δ, suggesting that additional electrostatic interactions in myo1c^IQ-tail help to stabilize binding. Remarkably, high concentrations of soluble inositol phosphates induce dissociation of myo1c^IQ-tail from LUVs, suggesting that phosphoinositides are able to bind to and dissociate from myo1c^IQ-tail as it remains bound to the membrane.Myosin-I isoforms are low molecular weight members of the myosin superfamily that link cell membranes with the actin cytoskeleton and play crucial roles driving a diverse array of dynamic membrane processes (1–5). Cell biological studies have shown that myosin-I isoforms localize and fractionate with cell membranes (2, 6), and biochemical experiments have shown myosin-I isoforms bind directly to lipid membranes (7–10). Thus, a key property of some myosin-I isoforms is their ability to bind membranes.myo1c is a widely expressed vertebrate myosin-I isoform that has roles in a variety of important membrane events, including insulin-stimulated fusion of vesicles containing glucose transporter-4 with the plasma membrane (2, 11), compensatory endocytosis following regulated exocytosis (12), and tensioning of mechano-sensitive ion channels (3). The mechanisms of myo1c targeting and anchoring to specific regions on the membrane to support these functions are not well understood. However, evidence is building that myo1c targeting requires direct binding of myo1c to phosphoinositides in cell membranes (13–16).We have shown that binding of myo1c to membranes is mediated by a putative pleckstrin homology (PH)³ domain in its tail that binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) and other phosphoinositides with high affinity (K_d <0.5 μm in terms of accessible phosphoinositide concentration) (13). myo1c also binds soluble inositol phosphates (e.g. inositol 1,4,5-trisphosphate (InsP₃)) with similar affinity. Point mutations of amino acids known to be essential for phosphoinositide binding in other PH domains inhibit myo1c binding to PtdIns(4,5)P₂ in vitro, and these mutations disrupt membrane localization in vivo (13). The affinity of myo1c for PtdIns(4,5)P₂-containing membranes is increased by the presence of additional anionic phospholipids in the membrane. This increased affinity may be due to nonspecific electrostatic interactions between the anionic phospholipids and positively charged regions within the myo1c tail or regulatory domain (13, 17), which is similar to what has been found for the guanine nucleotide exchange factor, ARNO (18). However, high affinity membrane binding via these nonspecific electrostatic interactions (i.e. binding in the absence of PtdIns(4,5)P₂) requires the membrane composition to contain a nonphysiological mole fraction (e.g. >40% phosphatidylserine) of anionic phospholipids (13, 14).Because phosphoinositide binding is important for the cellular localization and function of myo1c (13), it is important to determine the physical constants that define this interaction. Determining the kinetics of membrane attachment will provide insight into the relationship between membrane attachment and actin attachment lifetimes and will also provide details about the role of anionic lipids in regulating membrane attachment. Therefore, we used stopped-flow kinetics to measure the in vitro association and dissociation kinetics of myo1c from LUVs as a function of phosphoinositide composition and anionic charge.     

Phosphatidylinositol 3-Phosphate 5-Kinase,FAB1/PIKfyve Kinase Mediates Endosome Maturation to Establish Endosome-Cortical Microtubule Interaction in Arabidopsis

Phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂] is an important lipid in membrane trafficking in animal and yeast systems; however, its role is still largely obscure in plants. Here, we demonstrate that the phosphatidylinositol 3-phosphate 5-kinase, formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), and its product, PtdIns(3,5)P₂, are essential for the maturation process of endosomes to mediate cortical microtubule association of endosomes, thereby controlling proper PIN-FORMED protein trafficking in young cortical and stele cells of root. We found that FAB1 predominantly localizes on the Sorting Nexin1 (SNX1)-residing late endosomes, and a loss of FAB1 function causes the release of late endosomal proteins, Ara7, and SNX1 from the endosome membrane, indicating that FAB1, or its product PtdIns(3,5)P₂, mediates the maturation process of the late endosomes. We also found that loss of FAB1 function causes the release of endosomes from cortical microtubules and disturbs proper cortical microtubule organization.Phosphoinositides play an important role in various cellular processes, including determination of organelle identity and mediating signal transduction by recruiting effector molecules to various organelles (Balla, 2013). Among those, D3-phosphorylated phosphoinositides, phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate [PtdIns(3,5)P₂], play essential roles in the endosomal trafficking and the vacuolar sorting. PtdIns3P is produced from phosphatidylinositol by class III PI3-kinase, vacuolar protein sorting34 (VPS34). In animal cells, PtdIns3P predominantly localizes to the early endosomes and controls endosome maturation, recycling, and degradation of cargo proteins coordinated with Rab5 GTPases (Jean and Kiger, 2012). In Arabidopsis (Arabidopsis thaliana), PtdIns3P mainly resides on the late endosomes and the prevacuolar membrane (Vermeer et al., 2006; Simon et al., 2014). Dysfunction of AtVPS34 resulted in a defect in growth (Welters et al., 1994), root hair elongation (Lee et al., 2008a), and pollen development (Lee et al., 2008b), indicating an important role for AtVPS34 and its product PtdIns3P in plant development. VPS34-mediated PtdIns3P synthesis at the endosomes recruits phosphatidylinositol 3-phosphate 5-kinase formation of aploid and binucleate cells1 (FAB1)/FYVE finger-containing phosphoinositide kinase (PIKfyve), then FAB1/PIKfyve produces PtdIns(3,5)P₂ from PtdIns3P to mediate late endosome maturation in yeast (Saccharomyces cerevisiae) and animals (Ho et al., 2012; Jean and Kiger, 2012). PtdIns(3,5)P₂ has crucial roles in the maintenance of lysosome/vacuole morphology and acidification, membrane trafficking of proteins, autophagy, and signaling mediation in response to various stresses (Shisheva, 2008).FAB1 was discovered in yeast, where mutations were found to result in the formation of aploid and binucleate cells (hence its name FAB). In addition, a loss of Fab1p function causes defects in vacuole function and morphology, cell surface integrity, and cell growth (Yamamoto et al., 1995). In mammalian cells, this kinase is called PIKfyve (FYVE is a PI3P-binding domain). FAB1/PIKfyve forms a protein complex with an adaptor-like protein, Vacuole14 (Bonangelino et al., 1997) and PtdIns(3,5)P₂ 5-phosphatase (Fig. 4; Gary et al., 2002), indicating that the FAB1 complex catalyzes both PtdIns(3,5)P₂ synthesis and turnover simultaneously. In mammalian cells, interference of FAB1/PIKfyve function causes severe defects during embryogenesis, resulting in embryonic lethality in Drosophila spp., Caenorhabditis elegans, and mice (Nicot et al., 2006; Rusten et al., 2006; Ikonomov et al., 2011; Takasuga et al., 2013). Whereas most genomes from human to yeast contain a single-copy gene, the Arabidopsis genome codes for four FAB1 genes (FAB1A–D), of which only FAB1A and FAB1B contain a FYVE domain (Mueller-Roeber and Pical, 2002), and fab1a/fab1b double mutant reveals male gametophyte lethality phenotype in Arabidopsis (Whitley et al., 2009). The mutant pollen shows severe defects in vacuolar reorganization following the first mitotic division of development, suggesting an important role of FAB1 and PtdIns(3,5)P₂ in vacuolar rearrangement for pollen development (Whitley et al., 2009).Open in a separate windowFigure 4.Localization of endosomal markers upon down-regulation of FAB1A/B or inhibition of PtdIns(3,5)P₂ synthesis in young root cortical cells. Localization of mRFP-SYP43, mRFP-vesicle-associated membrane protein (VAMP727), mRFP-ARA7, and SNX1-mRFP without estradiol (A, E, I, and M) or with estradiol (B, F, J, and N) in the FAB1A/B-amiRNA line, or wild-type (WT) plants without YM201636 (C, G, K, and O) or with YM201636 (D, H, L, and P). Bar = 10 μm. Measurement of fluorescent dot structures (Q). Data represent fluorescent dots per cell (mean ± sd). *, P < 0.001 (Student’s t test).We previously developed a transgenic Arabidopsis line that is able to conditionally down-regulate FAB1A and FAB1B expression simultaneously, and demonstrated that a loss of FAB1 function causes various abnormal phenotypes, including growth inhibition, hypersensitivity to exogenous auxin, disturbance of root gravitropism, and floral organ abnormalities (Hirano et al., 2011). In addition, we found that down-regulation of FAB1A/B expression impaired endomembrane homeostasis, including endocytosis, vacuole formation, and vacuolar acidification, likely causing pleiotropic developmental phenotypes that mostly related to the auxin signaling in Arabidopsis (Hirano et al., 2011; Hirano and Sato, 2011). In plants, auxin is a crucial phytohormone that has a wide variety of physiological roles associated with growth, development, and tropic responses (Zhao, 2010). The polar cell-to-cell transport of auxin is mediated by auxin transporters localized on the plasma membrane (PM), such as PIN-FORMED (PIN) proteins (Vieten et al., 2007; Feraru and Friml, 2008). PINs are used as model molecules for polarity establishment on the PM in Arabidopsis. The establishment of PIN polarity is accomplished by the recycling of PINs between the PM and endosomal compartments comprising the trans-Golgi network/early endosomes (TGN/EEs) and the late endosomes (LEs)/prevacuolar compartments. The PIN-recycling pathway is mediated by multiple endosomal regulatory proteins, such as Rab family GTPases and Sorting Nexin (SNX; Jaillais et al., 2006; Park and Jürgens, 2011).Rab proteins function as molecular switches to regulate the tethering and fusion step of transport vesicles to target membranes. Rab5 members of the Rab GTPases have various functions in the endocytic pathway in eukaryotes. The maturation of the early-to-late endosomes is regulated by Rab5-to-Rab7 conversion, which is regulated by the Mon1/Sand-1-Ccz1 complex (Nordmann et al., 2010; Poteryaev et al., 2010). In plants, Rab5-family proteins, Ara6 and Ara7, and Rha1 play important roles in Rab5-mediated endosomal trafficking including the vacuolar trafficking pathway, thereby regulating of the polar transport of auxin and responses to environmental conditions (Ebine et al., 2011; Inoue et al., 2013).SNXs are composed of two conserved domains: the PHOX domain, involved in the interaction with the phosphoinositides, PtdIns3P and PtdIns (3,5)P₂, in the endosomal membrane in animals (Cozier et al., 2002), and the BAR domain, mediating dimerization and binding to curved membranes (Peter et al., 2004). Loss of SNX function disrupts the stable association of the retromer subcomplex, VPS26-Vps29-Vps35, with endosomal membranes, and thus results in retromer dysfunction, indicating that SNXs have a crucial role in the assembly and maintenance of the core retromer function (Teasdale et al., 2001; Cullen and Korswagen, 2012). The first plant SNX was identified as a protein that interacts with various receptor kinases in Brassica oleracea (Vanoosthuyse et al., 2003), and then three SNX genes (SNX1, SNX2a, and SNX2b) were identified in Arabidopsis. The snx1 null mutant exhibits a semidwarf phenotype with other subtle developmental defects (Pourcher et al., 2010). SNX1 is localized to the late endosome and is involved in PIN2 recycling between endosomes and the PM (Jaillais et al., 2006). SNX1 has been reported to interact with cortical microtubules via the microtubule-associated protein Cytoplasmic Linker Associated Protein (CLASP), and the clasp1 null mutant displays aberrant SNX1 endosomes and enhanced PIN2 degradation in the lytic vacuoles, suggesting that an association of SNX1 endosomes and CLASP is important for recycling of PIN transporters (Ambrose et al., 2013).Although many analyses of FAB1/PIKfyve, Rab5 family GTPases, SNXs, and microtubles have been reported, and there are significant similarities in endosomal trafficking, a functional relationship between them is still largely obscure.In this study, we demonstrate that FAB1 produced PtdIns(3,5)P₂ in Arabidopsis, and knockdown of FAB1 expression or inhibition of FAB1 activity with a FAB1/PIKfyve inhibitor, YM201636, decreased PtdIns(3,5)P₂ content. We also found that FAB1 and its product PtdIns(3,5)P₂ mediate the late endosome maturation by recruiting endosomal effector molecules, Ara7 and SNX1, onto endosomes to establish endosome-cortical microtubule interaction. Subsequently, the basal polarity of PIN2 in young cortical cells and PIN1 in stele cells is achieved.     

Differential Regulation of Cell Type-specific Apoptosis by Stromelysin-3: A POTENTIAL MECHANISM VIA THE CLEAVAGE OF THE LAMININ RECEPTOR DURING TAIL RESORPTION IN XENOPUS LAEVIS*

Matrix metalloproteinases (MMPs) have been extensively studied because of their functional attributes in development and diseases. However, relatively few in vivo functional studies have been reported on the roles of MMPs in postembryonic organ development. Amphibian metamorphosis is a unique model for studying MMP function during vertebrate development because of its dependence on thyroid hormone (T3) and the ability to easily manipulate this process with exogenous T3. The MMP stromelysin-3 (ST3) is induced by T3, and its expression correlates with cell death during metamorphosis. We have previously shown that ST3 is both necessary and sufficient for larval epithelial cell death in the remodeling intestine. To investigate the roles of ST3 in other organs and especially on different cell types, we have analyzed the effect of transgenic overexpression of ST3 in the tail of premetamorphic tadpoles. We report for the first time that ST3 expression, in the absence of T3, caused significant muscle cell death in the tail of premetamorphic transgenic tadpoles. On the other hand, only relatively low levels of epidermal cell death were induced by precocious ST3 expression in the tail, contrasting what takes place during natural and T3-induced metamorphosis when ST3 expression is high. This cell type-specific apoptotic response to ST3 in the tail suggests distinct mechanisms regulating cell death in different tissues. Furthermore, our analyses of laminin receptor, an in vivo substrate of ST3 in the intestine, suggest that laminin receptor cleavage may be an underlying mechanism for the cell type-specific effects of ST3.The extracellular matrix (ECM),³ the dynamic milieu of the cell microenvironment, plays a critical role in dictating the fate of the cell. The cross-talk between the cell and ECM and the timely catabolism of the ECM are crucial for tissue remodeling during development (1). Matrix metalloproteinases (MMPs), extrinsic proteolytic regulators of the ECM, mediate this process to a large extent. MMPs are a large family of Zn²⁺-dependent endopeptidases potentially capable of cleaving the extracellular as well as nonextracellular proteins (2–9). The MMP superfamily includes collagenases, gelatinases, stromelysins, and membrane-type MMPs based on substrate specificity and domain organization (2–4). MMPs have been implicated to influence a wide range of physiological and pathological processes (10–13). The roles of MMPs appear to be very complex. For example, MMPs have been suggested to play roles in both tumor promotion and suppression (13–19). Unfortunately, relatively few functional studies have been carried out in vivo, especially in relation to the mechanisms involved during vertebrate development.Amphibian metamorphosis presents a fascinating experimental model to study MMP function during postembryonic development. A unique and salient feature of the metamorphic process is the absolute dependence on the signaling of thyroid hormone (20–23). This makes it possible to prevent metamorphosis by simply inhibiting the synthesis of endogenous T3 or to induce precocious metamorphosis by merely adding physiological levels of T3 in the rearing water of premetamorphic tadpoles. Gene expression screens have identified the MMP stromelysin-3 (ST3) as a direct T3 response gene (24–27). Expression studies have revealed a distinct spatial and temporal ST3 expression profile in correlation with metamorphic event, especially cell death (25, 28–31). Organ culture studies on intestinal remodeling have directly substantiated an essential role of ST3 in larval epithelial cell death and ECM remodeling (32). Furthermore, precocious expression of ST3 alone in premetamorphic tadpoles through transgenesis is sufficient to induce ECM remodeling and larval epithelial apoptosis in the tadpole intestine (33). Thus, ST3 appears to be necessary and sufficient for intestinal epithelial cell death during metamorphosis.ST3 was first isolated as a breast cancer-associated gene (34), and unlike most other MMPs, ST3 is secreted as an active protease through a furin-dependent intracellular activation mechanism (35). Like many other MMPs, ST3 is expressed in a number of pathological processes, including most human carcinomas (11, 36–40), as well as in many developmental processes in mammals (10, 34, 41–43), although the physiological and pathological roles of ST3 in vivo are largely unknown in mammals. Interestingly, compared with other MMPs, ST3 has only weak activities toward ECM proteins in vitro but stronger activities against non-ECM proteins like α1 proteinase inhibitor and IGFBP-1 (44–46). Although ST3 may cleave ECM proteins strongly in the in vivo environment, these findings suggest that the cleavage of non-ECM proteins is likely important for its biological roles. Consistently, we have recently identified a cell surface receptor, laminin receptor (LR) as an in vivo substrate of ST3 in the tadpole intestine during metamorphosis (47–49). Analyses of LR expression and cleavage suggest that LR cleavage by ST3 is likely an important mechanism by which ST3 regulates the interaction between the larval epithelial cells and the ECM to induce cell death during intestinal remodeling (47, 48).Here, to investigate the role of ST3 in the apoptosis in other tissues during metamorphosis and whether LR cleavage serves as a mechanism for ST3 to regulate the fate of different cell types, we have analyzed the effects of precocious expression of ST3 in premetamorphic tadpole tail. The tail offers an opportunity to examine the effects of ST3 on different cell types. The epidermis, the fast and slow muscles, and the connective tissue underlying the epidermis in the myotendinous junctions and surrounding the notochord constitute the major tissue types in tail (50). Even though death is the destiny of all these cell types, it is not clear whether they all die through similar or different mechanisms. Microscopic and histochemical analyses have shown that at least the muscle and epidermal cells undergo T3-dependent apoptosis during metamorphosis (23, 29, 51, 52). To study whether ST3 regulates apoptosis of these two cell types, we have made use of the transgenic animals that express a transgenic ST3 under the control of a heat shock-inducible promoter (33). We show that whereas extensive apoptosis is present in both the epidermis and muscles during natural as well as T3-induced metamorphosis, transgenic expression of ST3 induces cell death predominantly in the muscles. Furthermore, we show that LR is expressed in the epidermis and connective tissue but not in muscles of the tadpole tail. More importantly, LR cleavage products are present in the tail during natural metamorphosis but not in transgenic tadpoles overexpressing ST3. These results suggest that ST3 has distinct effects on the epidermis and muscles in the tail, possibly because of the tissue-specific expression and function of LR.     

Potentiation of Growth Factor Signaling by Insulin-like Growth Factor-binding Protein-3 in Breast Epithelial Cells Requires Sphingosine Kinase Activity

We have investigated the mechanism underlying potentiation of epidermal growth factor receptor (EGFR) and type 1 insulin-like growth factor receptor (IGFR1) signaling by IGF-binding protein-3 (IGFBP-3) in MCF-10A breast epithelial cells, focusing on a possible involvement of the sphingosine kinase (SphK) system. IGFBP-3 potentiated EGF-stimulated EGF receptor activation and DNA synthesis, and this was blocked by inhibitors of SphK activity or small interference RNA-mediated silencing of SphK1, but not SphK2, expression. Similarly, IGFR1 phosphorylation and DNA synthesis stimulated by LR3-IGF-I (an IGF-I analog not bound by IGFBP-3), were enhanced by IGFBP-3, and this was blocked by SphK1 silencing. SphK1 expression and activity were stimulated by IGFBP-3 ∼2-fold over 24 h. Silencing of sphingosine 1-phosphate receptor 1 (S1P₁) or S1P₃, but not S1P₂, abolished the effect of IGFBP-3 on EGF-stimulated EGFR activation. The effects of IGFBP-3 could be reproduced with exogenous S1P or medium conditioned by cells treated with IGFBP-3, and this was also blocked by inhibition of S1P₁ and S1P₃. These data indicate that potentiation of growth factor signaling by IGFBP-3 in MCF-10A cells requires SphK1 activity and S1P₁/S1P₃, suggesting that S1P, the product of SphK activity and ligand for S1P₁ and S1P₃, is the “missing link” mediating IGF and EGFR transactivation and cell growth stimulation by IGFBP-3.Insulin-like growth factor-binding protein-3 (IGFBP-3)² is one of the family of six IGFBPs that bind the peptide growth factors IGF-I and IGF-II with high affinity and regulate their bioactivity (1). As the predominant carrier of IGFs in the endocrine system, IGFBP-3 regulates the movement of these growth factors from the circulation to target tissues and inhibits their proliferative and antiapoptotic cellular effects by blocking their activation of the type 1 IGF receptor (IGFR1) at the cell surface. In vitro studies in a variety of cell types have revealed that IGFBP-3 may also impact on cell growth and survival independently of modulating IGF bioactivity, inducing cell cycle arrest and apoptosis by regulation of apoptotic effector proteins (2–4) and interaction with nuclear receptors (5–7).There is, however, also evidence of an association between IGFBP-3 and enhanced cell proliferation. Some clinical studies in breast, prostate, pancreatic, renal cell, and non-small cell lung cancers have shown that a high level of tissue expression of IGFBP-3 correlates with increased tumor growth or malignancy (8–13). Although the mechanism linking IGFBP-3 with growth stimulation in vivo remains unclear, we and others have shown that, in vitro, IGFBP-3 can enhance the effects of stimulatory growth factors. Human and bovine skin fibroblasts exposed to low concentrations of exogenous IGFBP-3 exhibit enhanced IGF-stimulated DNA synthesis (14, 15), and similarly, exogenous and endogenous IGFBP-3 enhanced the growth response to IGF-I in the MCF-7 breast cancer cell line (16). We have also shown previously that IGFBP-3 is inhibitory to DNA synthesis in MCF-10A breast epithelial cells in the absence of exogenous growth factors or serum (17), but is growth stimulatory in the presence of EGF in the same cell line (18). There is no evidence that potentiation of EGF or IGF bioactivity by IGFBP-3 requires direct interaction between IGFBP-3 and the growth factor receptors (15, 18), but the mechanism underlying the effects of IGFBP-3 on growth factor signaling has not been elucidated.Recently it was suggested that, in human umbilical vein endothelial cells, an antiapoptotic effect of IGFBP-3 is associated with increased expression and activity of sphingosine kinase 1 (SphK1), and formation of the bioactive sphingolipid sphingosine 1-phosphate (S1P) (19, 20). SphK1 has been shown to have a role in oncogenesis (21), and S1P, acting both as an intracellular second messenger and extracellularly through activation of specific S1P receptors, stimulates cell proliferation and survival (22). In addition to transducing S1P signaling, the G-protein-coupled S1P receptors have been implicated in signal amplification of a variety of growth factors receptors, including the EGF and platelet-derived growth factor receptors, via receptor transactivation (23, 24). In this study we investigated whether the sphingosine kinase system is involved in modulation of growth factor receptor signaling pathways by IGFBP-3 and demonstrate that SphK1 expression is stimulated by IGFBP-3 in MCF-10A cells, and its activity is required for potentiation of EGF and IGF-I signaling by IGFBP-3 in these cells.     

The Phox Domain of Sorting Nexin 5 Lacks Phosphatidylinositol 3-Phosphate (PtdIns(3)P) Specificity and Preferentially Binds to Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2)

Subcellular retrograde transport of cargo receptors from endosomes to the trans-Golgi network is critically involved in a broad range of physiological and pathological processes and highly regulated by a genetically conserved heteropentameric complex, termed retromer. Among the retromer components identified in mammals, sorting nexin 5 and 1 (SNX5; SNX1) have recently been found to interact, possibly controlling the membrane binding specificity of the complex. To elucidate how the unique sequence features of the SNX5 phox domain (SNX5-PX) influence retrograde transport, we have determined the SNX5-PX structure by NMR and x-ray crystallography at 1.5 Å resolution. Although the core fold of SNX5-PX resembles that of other known PX domains, we found novel structural features exclusive to SNX5-PX. It is most noteworthy that in SNX5-PX, a long helical hairpin is added to the core formed by a new α2′-helix and a much longer α3-helix. This results in a significantly altered overall shape of the protein. In addition, the unique double PXXP motif is tightly packed against the rest of the protein, rendering this part of the structure compact, occluding parts of the putative phosphatidylinositol (PtdIns) binding pocket. The PtdIns binding and specificity of SNX5-PX was evaluated by NMR titrations with eight different PtdIns and revealed that SNX5-PX preferentially and specifically binds to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂). The distinct structural and PtdIns binding characteristics of SNX5-PX impart specific properties on SNX5, influencing retromer-mediated regulation of retrograde trafficking of transmembrane cargo receptors.The early work on retromer revealed its role in the trafficking of cargo proteins between endosomes and the trans-Golgi network (TGN),² although recently, retromer involvement in many other physiological and developmental processes has been uncovered (1, 2). The best studied proteins associated with retromer activity are intracellular sorting receptors such as the yeast vacuolar protein-10 (Vps10) and mammalian mannose 6-phosphate receptors (3, 4). These receptors sort acid hydrolases, enzymes essential for protein degradation, out of the TGN into the yeast vacuole or the mammalian lysosome. Upon releasing their substrates, these cargos traffic back to the TGN to mediate further rounds of cargo-hydrolase transportation. Similar retrograde trafficking of cargo proteins involving signaling molecules such as Wnt and amyloid precursor protein (APP) are thought to be critical for their secretion and function (5, 6). Retrograde transportation is highly regulated by the heteropentameric retromer complex that consists of a sorting nexin (SNX) dimer (e.g. Vps5 and Vps17 in yeast) and a Vps26/29/35 trimer (7). In mammals, the binding of the SNX dimer to specific phosphatidylinositol (PtdIns) determines its subcellular membrane association and governs the recruitment of the Vps trimer to endosomal compartments. Mammalian orthologs of the trimer have been biochemically characterized, and their interaction and function in cargo protein trafficking is well established (8). More recently, crystal structures of three Vps proteins in the trimer suggested how this trimer interacts with the SNX dimer and cargo proteins as well as with curved membranes (9–12). In the SNX dimer, SNX1 and SNX2 are thought to be interchangeable Vps5 orthologs (13, 14). The NMR structure of SNX1 revealed details of PI(3)P specific binding, thereby explaining its role in endosomal trafficking (15). The identity for SNX5 as a potential functional mammalian ortholog of Vps17, however, was not revealed until recently.Although initially identified as a Fanconi anemia complementation group A (FANCA)-binding protein (16), SNX5 was later shown to play an important role in membrane trafficking (17–19). SNX5 contains a PX domain (SNX5-PX) that is the signature feature in defining the SNX family, composed of 30 members at present (20) (Fig. 1B). In addition, SNX5 possesses a C-terminal BAR (Bin/Amphiphysin/Rvs) domain that has been reported to interact with a number of other proteins involved in endosomal trafficking (17, 21–27). It functions as a dimerization module that senses and/or induces membrane curvature (28, 29). Our previous biochemical study suggested a specific interaction between SNX5 and SNX1 through which the two SNXs mutually influence each other''s effect in endosomal trafficking of epidermal growth factor receptor upon epidermal growth factor stimulation (17). In support of this observation are several recent reports that indicate a critical role of SNX5 and the closely related SNX6, beyond that of SNX1 and SNX2, on retrograde sorting of mannose 6-phosphate receptor (24, 27). Therefore, SNX5 and SNX6 may be functionally interchangeable orthologs of Vps17 in mammalian cells (7, 24). Furthermore, in contrast to some reports (18, 30), SNX5 partially localizes to late endosomes and the TGN, exhibiting very low binding affinity for PtdIns(3)P (17), the substrate for phox domain proteins associating with early endosome association. Therefore, the subcellular localization and function of the SNX dimer in SNX5 function may depend on its unique structure that is different from other known PX domains.Open in a separate windowFIGURE 1.Amino Acid sequence alignment of phox domains and domain architecture of the mammalian sorting nexin family. A, comparative sequence alignment of PX domains for residues equivalent to Gly⁴⁹–Leu¹¹⁹ of the p40-PX domain (adapted from Worby and Dixon (21)). Prolines in the Pro-X-X-Pro motif are highlighted in yellow, and residues involved in phospholipid binding in the p40-PX domain are boxed in magenta. Arg⁵⁸ and Arg¹⁰⁵ are marked with magenta triangles, and Tyr⁵⁹ and Lys⁹² are marked with black stars at the bottom of the sequences. The two conserved Arg residues and Lys⁹² of p40-PX in other PX domains are highlighted in dark blue boxes; those corresponding to Tyr⁵⁹ are boxed in green. The secondary structure elements of p40-PX are indicated by yellow arrows (β-sheets) and red ovals (α-helices). The three sequence stretches that are unique in SNX5-PX (or SNX6-PX) are enclosed in a bright blue box. B, domain architecture of SNX family members. The four classes within the SNX family are designated as PX SNXs, PX-BAR SNXs, SH3-PX-BAR, and PX-other domain SNXs. Each individual domain is depicted in a different color and/or shape. The following domains are depicted: PX (phox), BAR (Bin-Amphiphysin-Rvs), SH3 (Src homology 3), TM (transmembrane), PXA (PX domain-associated), RGS (regulator of G-protein signaling), MIT (microtubule interacting and trafficking), B41 (band 4.1 homology), TPR (tetratricopeptide repeat), PDZ (postsynaptic protein PSD-95/SAP90, the Drosophila melanogaster septate junction protein Discs-large, and the tight junction protein ZO-1), and RA (Ras association).Most PX domains of SNX family proteins preferentially bind PtdIns(3)P (30–34), with few exceptions that interact with other PtdIns (30, 32, 35). There are about a dozen structurally characterized PX domains from the SNX family or other PX domain-containing proteins currently deposited in the Protein Data Bank (PDB) data base. Their structures all share common core features, a three-stranded β-sheet that is abutted by three α-helices and an irregular strand containing the PXXP region. Analyses of the representative p47-PX and SNX3-PX domain structures suggested that PtdIns(3)P binding involves two conserved Arg residues at positions equivalent to Arg⁵⁸ and Arg¹⁰⁵ in p40-PX (36). Because equivalent Arg residues are found in the PX domains of most SNX family members, it is generally assumed that all SNX proteins interact with the PtdIns(3)P-enriched elements of the early endocytic compartments. The amino acid sequences of the PX domains of both SNX5 and SNX6, however, lack the two conserved Arg residues that are involved in PtdIns(3)P binding as well as comprising a ∼30-residue insertion immediately after the PXXP motif (Fig. 1A). In addition, the PXXP motif is extended into a double PXXP motif with the sequence PXXPXXP. These unique sequence features set SNX5/6 apart from the other SNX family members. In the p40-PX domain and yeast SNX3, the two conserved Arg residues, the loop between the PXXP motif, and the α3-helix are involved in forming the binding pocket for the phosphate groups of PtdIns(3)P (36, 37). Therefore, changes in length and sequence in this region in SNX5/6-PX are expected to have profound impact on the specific structure and conformation required for PtdIns recognition.To elucidate how its unique sequence features influence the function of SNX5 in retromer-mediated retrograde membrane trafficking, we structurally investigated the SNX5-PX domain by NMR spectroscopy and x-ray crystallography. Using direct NMR titrations, we established the PtdIns binding specificity of SNX5-PX. The high resolution (1.5 Å) crystal structure of the domain revealed its distinct features when compared with previously known family members. Our results demonstrate that the SNX5-PX domain is indeed unique, both with respect to its structure as well as with respect to ligand binding. These findings have important implications for the function of SNX5 in the subcellular membrane trafficking and retrograde sorting.     

Analysis of Free Energy Signals Arising from Nucleotide Hybridization Between rRNA and mRNA Sequences during Translation in Eubacteria

A decoding algorithm is tested that mechanistically models the progressive alignments that arise as the mRNA moves past the rRNA tail during translation elongation. Each of these alignments provides an opportunity for hybridization between the single-stranded, -terminal nucleotides of the 16S rRNA and the spatially accessible window of mRNA sequence, from which a free energy value can be calculated. Using this algorithm we show that a periodic, energetic pattern of frequency 1/3 is revealed. This periodic signal exists in the majority of coding regions of eubacterial genes, but not in the non-coding regions encoding the 16S and 23S rRNAs. Signal analysis reveals that the population of coding regions of each bacterial species has a mean phase that is correlated in a statistically significant way with species () content. These results suggest that the periodic signal could function as a synchronization signal for the maintenance of reading frame and that codon usage provides a mechanism for manipulation of signal phase.[1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32]     

Structure of Insoluble Rat Sperm Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH) via Heterotetramer Formation with Escherichia coli GAPDH Reveals Target for Contraceptive Design

Sperm glyceraldehyde-3-phosphate dehydrogenase has been shown to be a successful target for a non-hormonal contraceptive approach, but the agents tested to date have had unacceptable side effects. Obtaining the structure of the sperm-specific isoform to allow rational inhibitor design has therefore been a goal for a number of years but has proved intractable because of the insoluble nature of both native and recombinant protein. We have obtained soluble recombinant sperm glyceraldehyde-3-phosphate dehydrogenase as a heterotetramer with the Escherichia coli glyceraldehyde-3-phosphate dehydrogenase in a ratio of 1:3 and have solved the structure of the heterotetramer which we believe represents a novel strategy for structure determination of an insoluble protein. A structure was also obtained where glyceraldehyde 3-phosphate binds in the P_s pocket in the active site of the sperm enzyme subunit in the presence of NAD. Modeling and comparison of the structures of human somatic and sperm-specific glyceraldehyde-3-phosphate dehydrogenase revealed few differences at the active site and hence rebut the long presumed structural specificity of 3-chlorolactaldehyde for the sperm isoform. The contraceptive activity of α-chlorohydrin and its apparent specificity for the sperm isoform in vivo are likely to be due to differences in metabolism to 3-chlorolactaldehyde in spermatozoa and somatic cells. However, further detailed analysis of the sperm glyceraldehyde-3-phosphate dehydrogenase structure revealed sites in the enzyme that do show significant difference compared with published somatic glyceraldehyde-3-phosphate dehydrogenase structures that could be exploited by structure-based drug design to identify leads for novel male contraceptives.Glyceraldehyde-3-phosphate dehydrogenase-S (GAPDS³ in rat; GAPDH2 in human) is the sperm-specific isoform of GAPDH (1–3) and the sole GAPDH enzyme in sperm. GAPDS is highly conserved between species showing 94% identity between rat and mouse and 87% identity between rat and human. Within a particular species, GAPDS also shows significant sequence identity to its GAPDH paralogue, 70, 70, and 68% for rat, mouse, and human, respectively. The most striking difference between GAPDS and GAPDH is the presence of an N-terminal polyproline region in GAPDS, which is 97 residues in rat (accession number {"type":"entrez-nucleotide","attrs":{"text":"AJ297631","term_id":"9931190","term_text":"AJ297631"}}AJ297631), 105 in mouse (3), and 72 in human (2). GAPDS is restricted to the principal piece of the sperm flagellum (1, 2, 4) where it is localized to the fibrous sheath (5), an association proposed to be mediated via the N-terminal polyproline extension.GAPDS first came to prominence as a contraceptive target during the 1970s (6–8). Investigations showed that treatment of sperm with α-chlorohydrin or a number of related compounds could inhibit GAPDS activity (9–11), sperm motility (9–13), and the fertilization of oocytes in vitro (14). The metabolite of these compounds, 3-chlorolactaldehyde (15–17), selectively inhibited GAPDS, having no effect on the activity of somatic cell GAPDH (18, 19), providing the specificity required for a potential contraceptive. Questions surrounding these particular compounds were raised when a number of side effects were evident from in vivo trials (7, 20–22); however, the design of small molecule inhibitors of GAPDS may provide a viable alternative. Its potential as a contraceptive target was supported by data from mice where GAPDS^−/− males (23) were infertile because of defects in sperm motility.Glyceraldehyde-3-phosphate dehydrogenases are tetrameric enzymes that catalyze the oxidative phosphorylation of d-glyceraldehyde 3-phosphate (Glc-3-P) into 1,3-diphosphoglycerate in the presence of an NAD cofactor via a two-step chemical mechanism (24). The first models of substrate binding were proposed on the basis of crystal structures of the holoenzyme from lobster (25) and Bacillus stearothermophilus (26), and Moras and co-workers (25) identified two anion-binding sites postulated to correspond to those binding the C-3 phosphate group of d-Glc-3-P (P_s site) and the inorganic phosphate ion (P_i site).Structure-based design of small molecules to inhibit GAPDH is not unprecedented. GAPDH has been targeted from protozoan parasites (27–30), as the bloodstream forms rely solely on glycolysis for energy production (31, 32). A number of mammalian GAPDH structures have also been solved, including rabbit muscle (33, 34), human liver (35), and human placenta (36); however, no structures are available for sperm-specific isoforms of this enzyme.Active heterotetramers of GAPDH between different species have been reported and biochemically characterized previously, both in ratios of 2:2 and 3:1 (37–40). In this study we have successfully obtained crystals of rat recombinant GAPDS as a heterotetramer with Escherichia coli GAPDH in a 1:3 ratio. To understand the basis of inhibition of the sperm isoform by substrate analogue 3-chlorolactaldehyde, a metabolite of α-chlorohydrin, a structure was also determined in the presence of the substrate glyceraldehyde 3-phosphate. The sperm-specific structure was compared with the human placental GAPDH structure (PDB entry 1U8F; Ref. 36) to identify differences that may provide a target for the design of inhibitors specific to the GAPDS protein. The unique structural features identified offer potential candidates for further investigation as inhibitor targets.