Loss of PINK1 Function Promotes Mitophagy through Effects on Oxidative Stress and Mitochondrial Fission

Mitochondrial dysregulation is strongly implicated in Parkinson disease. Mutations in PTEN-induced kinase 1 (PINK1) are associated with familial parkinsonism and neuropsychiatric disorders. Although overexpressed PINK1 is neuroprotective, less is known about neuronal responses to loss of PINK1 function. We found that stable knockdown of PINK1 induced mitochondrial fragmentation and autophagy in SH-SY5Y cells, which was reversed by the reintroduction of an RNA interference (RNAi)-resistant plasmid for PINK1. Moreover, stable or transient overexpression of wild-type PINK1 increased mitochondrial interconnectivity and suppressed toxin-induced autophagy/mitophagy. Mitochondrial oxidant production played an essential role in triggering mitochondrial fragmentation and autophagy in PINK1 shRNA lines. Autophagy/mitophagy served a protective role in limiting cell death, and overexpressing Parkin further enhanced this protective mitophagic response. The dominant negative Drp1 mutant inhibited both fission and mitophagy in PINK1-deficient cells. Interestingly, RNAi knockdown of autophagy proteins Atg7 and LC3/Atg8 also decreased mitochondrial fragmentation without affecting oxidative stress, suggesting active involvement of autophagy in morphologic remodeling of mitochondria for clearance. To summarize, loss of PINK1 function elicits oxidative stress and mitochondrial turnover coordinated by the autophagic and fission/fusion machineries. Furthermore, PINK1 and Parkin may cooperate through different mechanisms to maintain mitochondrial homeostasis.Parkinson disease is an age-related neurodegenerative disease that affects ∼1% of the population worldwide. The causes of sporadic cases are unknown, although mitochondrial or oxidative toxins such as 1-methyl-4-phenylpyridinium, 6-hydroxydopamine (6-OHDA),³ and rotenone reproduce features of the disease in animal and cell culture models (1). Abnormalities in mitochondrial respiration and increased oxidative stress are observed in cells and tissues from parkinsonian patients (2, 3), which also exhibit increased mitochondrial autophagy (4). Furthermore, mutations in parkinsonian genes affect oxidative stress response pathways and mitochondrial homeostasis (5). Thus, disruption of mitochondrial homeostasis represents a major factor implicated in the pathogenesis of sporadic and inherited parkinsonian disorders (PD).The PARK6 locus involved in autosomal recessive and early-onset PD encodes for PTEN-induced kinase 1 (PINK1) (6, 7). PINK1 is a cytosolic and mitochondrially localized 581-amino acid serine/threonine kinase that possesses an N-terminal mitochondrial targeting sequence (6, 8). The primary sequence also includes a putative transmembrane domain important for orientation of the PINK1 domain (8), a conserved kinase domain homologous to calcium calmodulin kinases, and a C-terminal domain that regulates autophosphorylation activity (9, 10). Overexpression of wild-type PINK1, but not its PD-associated mutants, protects against several toxic insults in neuronal cells (6, 11, 12). Mitochondrial targeting is necessary for some (13) but not all of the neuroprotective effects of PINK1 (14), implicating involvement of cytoplasmic targets that modulate mitochondrial pathobiology (8). PINK1 catalytic activity is necessary for its neuroprotective role, because a kinase-deficient K219M substitution in the ATP binding pocket of PINK1 abrogates its ability to protect neurons (14). Although PINK1 mutations do not seem to impair mitochondrial targeting, PD-associated mutations differentially destabilize the protein, resulting in loss of neuroprotective activities (13, 15).Recent studies indicate that PINK1 and Parkin interact genetically (3, 16-18) to prevent oxidative stress (19, 20) and regulate mitochondrial morphology (21). Primary cells derived from PINK1 mutant patients exhibit mitochondrial fragmentation with disorganized cristae, recapitulated by RNA interference studies in HeLa cells (3).Mitochondria are degraded by macroautophagy, a process involving sequestration of cytoplasmic cargo into membranous autophagic vacuoles (AVs) for delivery to lysosomes (22, 23). Interestingly, mitochondrial fission accompanies autophagic neurodegeneration elicited by the PD neurotoxin 6-OHDA (24, 25). Moreover, mitochondrial fragmentation and increased autophagy are observed in neurodegenerative diseases including Alzheimer and Parkinson diseases (4, 26-28). Although inclusion of mitochondria in autophagosomes was once believed to be a random process, as observed during starvation, studies involving hypoxia, mitochondrial damage, apoptotic stimuli, or limiting amounts of aerobic substrates in facultative anaerobes support the concept of selective mitochondrial autophagy (mitophagy) (29, 30). In particular, mitochondrially localized kinases may play an important role in models involving oxidative mitochondrial injury (25, 31, 32).Autophagy is involved in the clearance of protein aggregates (33-35) and normal regulation of axonal-synaptic morphology (36). Chronic disruption of lysosomal function results in accumulation of subtly impaired mitochondria with decreased calcium buffering capacity (37), implicating an important role for autophagy in mitochondrial homeostasis (37, 38). Recently, Parkin, which complements the effects of PINK1 deficiency on mitochondrial morphology (3), was found to promote autophagy of depolarized mitochondria (39). Conversely, Beclin 1-independent autophagy/mitophagy contributes to cell death elicited by the PD toxins 1-methyl-4-phenylpyridinium and 6-OHDA (25, 28, 31, 32), causing neurite retraction in cells expressing a PD-linked mutation in leucine-rich repeat kinase 2 (40). Whereas properly regulated autophagy plays a homeostatic and neuroprotective role, excessive or incomplete autophagy creates a condition of “autophagic stress” that can contribute to neurodegeneration (28).As mitochondrial fragmentation (3) and increased mitochondrial autophagy (4) have been described in human cells or tissues of PD patients, we investigated whether or not the engineered loss of PINK1 function could recapitulate these observations in human neuronal cells (SH-SY5Y). Stable knockdown of endogenous PINK1 gave rise to mitochondrial fragmentation and increased autophagy and mitophagy, whereas stable or transient overexpression of PINK1 had the opposite effect. Autophagy/mitophagy was dependent upon increased mitochondrial oxidant production and activation of fission. The data indicate that PINK1 is important for the maintenance of mitochondrial networks, suggesting that coordinated regulation of mitochondrial dynamics and autophagy limits cell death associated with loss of PINK1 function.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Analysis of a Critical Interaction within the Archaeal Box C/D Small Ribonucleoprotein Complex

In archaea and eukarya, box C/D ribonucleoprotein (RNP) complexes are responsible for 2′-O-methylation of tRNAs and rRNAs. The archaeal box C/D small RNP complex requires a small RNA component (sRNA) possessing Watson-Crick complementarity to the target RNA along with three proteins: L7Ae, Nop5p, and fibrillarin. Transfer of a methyl group from S-adenosylmethionine to the target RNA is performed by fibrillarin, which by itself has no affinity for the sRNA-target duplex. Instead, it is targeted to the site of methylation through association with Nop5p, which in turn binds to the L7Ae-sRNA complex. To understand how Nop5p serves as a bridge between the targeting and catalytic functions of the box C/D small RNP complex, we have employed alanine scanning to evaluate the interaction between the Pyrococcus horikoshii Nop5p domain and an L7Ae box C/D RNA complex. From these data, we were able to construct an isolated RNA-binding domain (Nop-RBD) that folds correctly as demonstrated by x-ray crystallography and binds to the L7Ae box C/D RNA complex with near wild type affinity. These data demonstrate that the Nop-RBD is an autonomously folding and functional module important for protein assembly in a number of complexes centered on the L7Ae-kinkturn RNP.Many biological RNAs require extensive modification to attain full functionality in the cell (1). Currently there are over 100 known RNA modification types ranging from small functional group substitutions to the addition of large multi-cyclic ring structures (2). Transfer RNA, one of many functional RNAs targeted for modification (3-6), possesses the greatest modification type diversity, many of which are important for proper biological function (7). Ribosomal RNA, on the other hand, contains predominantly two types of modified nucleotides: pseudouridine and 2′-O-methylribose (8). The crystal structures of the ribosome suggest that these modifications are important for proper folding (9, 10) and structural stabilization (11) in vivo as evidenced by their strong tendency to localize to regions associated with function (8, 12, 13). These roles have been verified biochemically in a number of cases (14), whereas newly emerging functional modifications are continually being investigated.Box C/D ribonucleoprotein (RNP)³ complexes serve as RNA-guided site-specific 2′-O-methyltransferases in both archaea and eukaryotes (15, 16) where they are referred to as small RNP complexes and small nucleolar RNPs, respectively. Target RNA pairs with the sRNA guide sequence and is methylated at the 2′-hydroxyl group of the nucleotide five bases upstream of either the D or D′ box motif of the sRNA (Fig. 1, star) (17, 18). In archaea, the internal C′ and D′ motifs generally conform to a box C/D consensus sequence (19), and each sRNA contains two guide regions ∼12 nucleotides in length (20). The bipartite architecture of the RNP potentially enables the complex to methylate two distinct RNA targets (21) and has been shown to be essential for site-specific methylation (22).Open in a separate windowFIGURE 1.Organization of the archaeal box C/D complex. The protein components of this RNP are L7Ae, Nop5p, and fibrillarin, which together bind a box C/D sRNA. The regions of the Box C/D sRNA corresponding to the conserved C, D, C′, and D′ boxes are labeled. The target RNA binds the sRNA through Watson-Crick pairing and is methylated by fibrillarin at the fifth nucleotide from the D/D′ boxes (star).In addition to the sRNA, the archaeal box C/D complex requires three proteins for activity (23): the ribosomal protein L7Ae (24, 25), fibrillarin, and the Nop56/Nop58 homolog Nop5p (Fig. 1). L7Ae binds to both box C/D and the C′/D′ motifs (26), which respectively comprise kink-turn (27) or k-loop structures (28), to initiate the assembly of the RNP (29, 30). Fibrillarin performs the methyl group transfer from the cofactor S-adenosylmethionine to the target RNA (31-33). For this to occur, the active site of fibrillarin must be positioned precisely over the specific 2′-hydroxyl group to be methylated. Although fibrillarin methylates this functional group in the context of a Watson-Crick base-paired helix (guide/target), it has little to no binding affinity for double-stranded RNA or for the L7Ae-sRNA complex (22, 26, 33, 34). Nop5p serves as an intermediary protein bringing fibrillarin to the complex through its association with both the L7Ae-sRNA complex and fibrillarin (22). Along with its role as an intermediary between fibrillarin and the L7Ae-sRNA complex, Nop5p possesses other functions not yet fully understood. For example, Nop5p self-dimerizes through a coiled-coil domain (35) that in most archaea and eukaryotic homologs includes a small insertion sequence of unknown function (36, 37). However, dimerization and fibrillarin binding have been shown to be mutually exclusive in Methanocaldococcus jannaschii Nop5p, potentially because of the presence of this insertion sequence (36). Thus, whether Nop5p is a monomer or a dimer in the active RNP is still under debate.In this study, we focus our attention on the Nop5p protein to investigate its interaction with a L7Ae box C/D RNA complex because both the fibrillarin-Nop5p and the L7Ae box C/D RNA interfaces are known from crystal structures (29, 35, 38). Individual residues on the surface of a monomeric form of Nop5p (referred to as mNop5p) (22) were mutated to alanine, and the effect on binding affinity for a L7Ae box C/D motif RNA complex was assessed through the use of electrophoretic mobility shift assays. These data reveal that residues important for binding cluster within the highly conserved NOP domain (39, 40). To demonstrate that this domain is solely responsible for the affinity of Nop5p for the preassembled L7Ae box C/D RNA complex, we expressed and purified it in isolation from the full Nop5p protein. The isolated Nop-RBD domain binds to the L7Ae box C/D RNA complex with nearly wild type affinity, demonstrating that the Nop-RBD is truly an autonomously folding and functional module. Comparison of our data with the crystal structure of the homologous spliceosomal hPrp31-15.5K protein-U4 snRNA complex (41) suggests the adoption of a similar mode of binding, further supporting a crucial role for the NOP domain in RNP complex assembly.     

Specific Activation of mTORC1 by Rheb G-protein in Vitro Involves Enhanced Recruitment of Its Substrate Protein

Rheb G-protein plays critical roles in the TSC/Rheb/mTOR signaling pathway by activating mTORC1. The activation of mTORC1 by Rheb can be faithfully reproduced in vitro by using mTORC1 immunoprecipitated by the use of anti-raptor antibody from mammalian cells starved for nutrients. The low in vitro kinase activity against 4E-BP1 of this mTORC1 preparation is dramatically increased by the addition of recombinant Rheb. On the other hand, the addition of Rheb does not activate mTORC2 immunoprecipitated from mammalian cells by the use of anti-rictor antibody. The activation of mTORC1 is specific to Rheb, because other G-proteins such as KRas, RalA/B, and Cdc42 did not activate mTORC1. Both Rheb1 and Rheb2 activate mTORC1. In addition, the activation is dependent on the presence of bound GTP. We also find that the effector domain of Rheb is required for the mTORC1 activation. FKBP38, a recently proposed mediator of Rheb action, appears not to be involved in the Rheb-dependent activation of mTORC1 in vitro, because the preparation of mTORC1 that is devoid of FKBP38 is still activated by Rheb. The addition of Rheb results in a significant increase of binding of the substrate protein 4E-BP1 to mTORC1. PRAS40, a TOR signaling (TOS) motif-containing protein that competes with the binding of 4EBP1 to mTORC1, inhibits Rheb-induced activation of mTORC1. A preparation of mTORC1 that is devoid of raptor is not activated by Rheb. Rheb does not induce autophosphorylation of mTOR. These results suggest that Rheb induces alteration in the binding of 4E-BP1 with mTORC1 to regulate mTORC1 activation.Rheb defines a unique member of the Ras superfamily G-proteins (1). We have shown that Rheb proteins are conserved and are found from yeast to human (2). Although yeast and fruit fly have one Rheb, mouse and human have two Rheb proteins termed Rheb1 (or simply Rheb) and Rheb2 (RhebL1) (2). Structurally, these proteins contain G1-G5 boxes, short stretches of amino acids that define the function of the Ras superfamily G-proteins including guanine nucleotide binding (1, 3, 4). Rheb proteins have a conserved arginine at residue 15 that corresponds to residue 12 of Ras (1). The effector domain required for the binding with downstream effectors encompasses the G2 box and its adjacent sequences (1, 5). Structural analysis by x-ray crystallography further shows that the effector domain is exposed to solvent, is located close to the phosphates of GTP especially at residues 35–38, and undergoes conformational change during GTP/GDP exchange (6). In addition, all Rheb proteins end with the CAAX (C is cysteine, A is an aliphatic amino acid, and X is the C-terminal amino acid) motif that signals farnesylation. In fact, we as well as others have shown that these proteins are farnesylated (7–9).Rheb plays critical roles in the TSC/Rheb/mTOR signaling, a signaling pathway that plays central roles in regulating protein synthesis and growth in response to nutrient, energy, and growth conditions (10–14). Rheb is down-regulated by a TSC1·TSC2 complex that acts as a GTPase-activating protein for Rheb (15–19). Recent studies established that the GAP domain of TSC2 defines the functional domain for the down-regulation of Rheb (20). Mutations in the Tsc1 or Tsc2 gene lead to tuberous sclerosis whose symptoms include the appearance of benign tumors called hamartomas at different parts of the body as well as neurological symptoms (21, 22). Overexpression of Rheb results in constitutive activation of mTOR even in the absence of nutrients (15, 16). Two mTOR complexes, mTORC1 and mTORC2, have been identified (23, 24). Whereas mTORC1 is involved in protein synthesis activation mediated by S6K and 4EBP1, mTORC2 is involved in the phosphorylation of Akt in response to insulin. It has been suggested that Rheb is involved in the activation of mTORC1 but not mTORC2 (25).Although Rheb is clearly involved in the activation of mTOR, the mechanism of activation has not been established. We as well as others have suggested a model that involves the interaction of Rheb with the TOR complex (26–28). Rheb activation of mTOR kinase activity using immunoprecipitated mTORC1 was reported (29). Rheb has been shown to interact with mTOR (27, 30), and this may involve direct interaction of Rheb with the kinase domain of mTOR (27). However, this Rheb/mTOR interaction is a weak interaction and is not dependent on the presence of GTP bound to Rheb (27, 28). Recently, a different model proposing that FKBP38 (FK506-binding protein 38) mediates the activation of mTORC1 by Rheb was proposed (31, 32). In this model, FKBP38 binds mTOR and negatively regulates mTOR activity, and this negative regulation is blocked by the binding of Rheb to FKBP38. However, recent reports dispute this idea (33).To further characterize Rheb activation of mTOR, we have utilized an in vitro system that reproduces activation of mTORC1 by the addition of recombinant Rheb. We used mTORC1 immunoprecipitated from nutrient-starved cells using anti-raptor antibody and have shown that its kinase activity against 4E-BP1 is dramatically increased by the addition of recombinant Rheb. Importantly, the activation of mTORC1 is specific to Rheb and is dependent on the presence of bound GTP as well as an intact effector domain. FKBP38 is not detected in our preparation and further investigation suggests that FKBP38 is not an essential component for the activation of mTORC1 by Rheb. Our study revealed that Rheb enhances the binding of a substrate 4E-BP1 with mTORC1 rather than increasing the kinase activity of mTOR.     

Mapping Daunorubicin-binding Sites in the ATP-binding Cassette Transporter MsbA Using Site-specific Quenching by Spin Labels

ATP-binding cassette (ABC) transporters transduce the free energy of ATP hydrolysis to power the mechanical work of substrate translocation across cell membranes. MsbA is an ABC transporter implicated in trafficking lipid A across the inner membrane of Escherichia coli. It has sequence similarity and overlapping substrate specificity with multidrug ABC transporters that export cytotoxic molecules in humans and prokaryotes. Despite rapid advances in structure determination of ABC efflux transporters, little is known regarding the location of substrate-binding sites in the transmembrane segment and the translocation pathway across the membrane. In this study, we have mapped residues proximal to the daunorubicin (DNR)-binding site in MsbA using site-specific, ATP-dependent quenching of DNR intrinsic fluorescence by spin labels. In the nucleotide-free MsbA intermediate, DNR-binding residues cluster at the cytoplasmic end of helices 3 and 6 at a site accessible from the membrane/water interface and extending into an aqueous chamber formed at the interface between the two transmembrane domains. Binding of a nonhydrolyzable ATP analog inverts the transporter to an outward-facing conformation and relieves DNR quenching by spin labels suggesting DNR exclusion from proximity to the spin labels. The simplest model consistent with our data has DNR entering near an elbow helix parallel to the water/membrane interface, partitioning into the open chamber, and then translocating toward the periplasm upon ATP binding.ATP-binding cassette (ABC)² transporters transduce the energy of ATP hydrolysis to power the movement of a wide range of substrates across the cell membranes (1, 2). They constitute the largest family of prokaryotic transporters, import essential cell nutrients, flip lipids, and export toxic molecules (3). Forty eight human ABC transporters have been identified, including ABCB1, or P-glycoprotein, which is implicated in cross-resistance to drugs and cytotoxic molecules (4, 5). Inherited mutations in these proteins are linked to diseases such as cystic fibrosis, persistent hypoglycemia of infancy, and immune deficiency (6).The functional unit of an ABC transporter consists of four modules. Two highly conserved ABCs or nucleotide-binding domains (NBDs) bind and hydrolyze ATP to supply the active energy for transport (7). ABCs drive the mechanical work of proteins with diverse functions ranging from membrane transport to DNA repair (3, 5). Substrate specificity is determined by two transmembrane domains (TMDs) that also provide the translocation pathway across the bilayer (7). Bacterial ABC exporters are expressed as monomers, each consisting of one NBD and one TMD, that dimerize to form the active transporter (3). The number of transmembrane helices and their organization differ significantly between ABC importers and exporters reflecting the divergent structural and chemical nature of their substrates (1, 8, 9). Furthermore, ABC exporters bind substrates directly from the cytoplasm or bilayer inner leaflet and release them to the periplasm or bilayer outer leaflet (10, 11). In contrast, bacterial importers have their substrates delivered to the TMD by a dedicated high affinity substrate-binding protein (12).In Gram-negative bacteria, lipid A trafficking from its synthesis site on the inner membrane to its final destination in the outer membrane requires the ABC transporter MsbA (13). Although MsbA has not been directly shown to transport lipid A, suppression of MsbA activity leads to cytoplasmic accumulation of lipid A and inhibits bacterial growth strongly suggesting a role in translocation (14-16). In addition to this role in lipid A transport, MsbA shares sequence similarity with multidrug ABC transporters such as human ABCB1, LmrA of Lactococcus lactis, and Sav1866 of Staphylococcus aureus (16-19). ABCB1, a prototype of the ABC family, is a plasma membrane protein whose overexpression provides resistance to chemotherapeutic agents in cancer cells (1). LmrA and MsbA have overlapping substrate specificity with ABCB1 suggesting that both proteins can function as drug exporters (18, 20). Indeed, cells expressing MsbA confer resistance to erythromycin and ethidium bromide (21). MsbA can be photolabeled with the ABCB1/LmrA substrate azidopine and can transport Hoechst 33342 ({"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342) across membrane vesicles in an energy-dependent manner (21).The structural mechanics of ABC exporters was revealed from comparison of the MsbA crystal structures in the apo- and nucleotide-bound states as well as from analysis by spin labeling EPR spectroscopy in liposomes (17, 19, 22, 23). The energy harnessed from ATP binding and hydrolysis drives a cycle of NBD association and dissociation that is transmitted to induce reorientation of the TMD from an inward- to outward-facing conformation (17, 19, 22). Large amplitude motion closes the cytoplasmic end of a chamber found at the interface between the two TMDs and opens it to the periplasm (23). These rearrangements lead to significant changes in chamber hydration, which may drive substrate translocation (22).Substrate binding must precede energy input, otherwise the cycle is futile, wasting the energy of ATP hydrolysis without substrate extrusion (7). Consistent with this model, ATP binding reduces ABCB1 substrate affinity, potentially through binding site occlusion (24-26). Furthermore, the TMD substrate-binding event signals the NBD to stimulate ATP hydrolysis increasing transport efficiency (1, 27, 28). However, there is a paucity of information regarding the location of substrate binding, the transport pathway, and the structural basis of substrate recognition by ABC exporters. In vitro studies of MsbA substrate specificity identify a broad range of substrates that stimulate ATPase activity (29). In addition to the putative physiological substrates lipid A and lipopolysaccharide (LPS), the ABCB1 substrates Ilmofosine, {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342, and verapamil differentially enhance ATP hydrolysis of MsbA (29, 30). Intrinsic MsbA tryptophan (Trp) fluorescence quenching by these putative substrate molecules provides further support of interaction (29).Extensive biochemical analysis of ABCB1 and LmrA provides a general model of substrate binding to ABC efflux exporters. This so-called “hydrophobic cleaner model” describes substrates binding from the inner leaflet of the bilayer and then translocating through the TMD (10, 31, 32). These studies also identified a large number of residues involved in substrate binding and selectivity (33). When these crucial residues are mapped onto the crystal structures of MsbA, a subset of homologous residues clusters to helices 3 and 6 lining the putative substrate pathway (34). Consistent with a role in substrate binding and specificity, simultaneous replacement of two serines (Ser-289 and Ser-290) in helix 6 of MsbA reduces binding and transport of ethidium and taxol, although {"type":"entrez-nucleotide","attrs":{"text":"H33342","term_id":"978759","term_text":"H33342"}}H33342 and erythromycin interactions remain unaffected (34).The tendency of lipophilic substrates to partition into membranes confounds direct analysis of substrate interactions with ABC exporters (35, 36). Such partitioning may promote dynamic collisions with exposed Trp residues and nonspecific cross-linking in photo-affinity labeling experiments. In this study, we utilize a site-specific quenching approach to identify residues in the vicinity of the daunorubicin (DNR)-binding site (37). Although the data on DNR stimulation of ATP hydrolysis is inconclusive (20, 29, 30), the quenching of MsbA Trp fluorescence suggests a specific interaction. Spin labels were introduced along transmembrane helices 3, 4, and 6 of MsbA to assess their ATP-dependent quenching of DNR fluorescence. Residues that quench DNR cluster along the cytoplasmic end of helices 3 and 6 consistent with specific binding of DNR. Furthermore, many of these residues are not lipid-exposed but face the putative substrate chamber formed between the two TMDs. These residues are proximal to two Trps, which likely explains the previously reported quenching (29). Our results suggest DNR partitions to the membrane and then binds MsbA in a manner consistent with the hydrophobic cleaner model. Interpretation in the context of the crystal structures of MsbA identifies a putative translocation pathway through the transmembrane segment.     

Intersectin Regulates Dendritic Spine Development and Somatodendritic Endocytosis but Not Synaptic Vesicle Recycling in Hippocampal Neurons

Intersectin-short (intersectin-s) is a multimodule scaffolding protein functioning in constitutive and regulated forms of endocytosis in non-neuronal cells and in synaptic vesicle (SV) recycling at the neuromuscular junction of Drosophila and Caenorhabditis elegans. In vertebrates, alternative splicing generates a second isoform, intersectin-long (intersectin-l), that contains additional modular domains providing a guanine nucleotide exchange factor activity for Cdc42. In mammals, intersectin-s is expressed in multiple tissues and cells, including glia, but excluded from neurons, whereas intersectin-l is a neuron-specific isoform. Thus, intersectin-I may regulate multiple forms of endocytosis in mammalian neurons, including SV endocytosis. We now report, however, that intersectin-l is localized to somatodendritic regions of cultured hippocampal neurons, with some juxtanuclear accumulation, but is excluded from synaptophysin-labeled axon terminals. Consistently, intersectin-l knockdown (KD) does not affect SV recycling. Instead intersectin-l co-localizes with clathrin heavy chain and adaptor protein 2 in the somatodendritic region of neurons, and its KD reduces the rate of transferrin endocytosis. The protein also co-localizes with F-actin at dendritic spines, and intersectin-l KD disrupts spine maturation during development. Our data indicate that intersectin-l is indeed an important regulator of constitutive endocytosis and neuronal development but that it is not a prominent player in the regulated endocytosis of SVs.Clathrin-mediated endocytosis (CME)⁴ is a major mechanism by which cells take up nutrients, control the surface levels of multiple proteins, including ion channels and transporters, and regulate the coupling of signaling receptors to downstream signaling cascades (1-5). In neurons, CME takes on additional specialized roles; it is an important process regulating synaptic vesicle (SV) availability through endocytosis and recycling of SV membranes (6, 7), it shapes synaptic plasticity (8-10), and it is crucial in maintaining synaptic membranes and membrane structure (11).Numerous endocytic accessory proteins participate in CME, interacting with each other and with core components of the endocytic machinery such as clathrin heavy chain (CHC) and adaptor protein-2 (AP-2) through specific modules and peptide motifs (12). One such module is the Eps15 homology domain that binds to proteins bearing NPF motifs (13, 14). Another is the Src homology 3 (SH3) domain, which binds to proline-rich domains in protein partners (15). Intersectin is a multimodule scaffolding protein that interacts with a wide range of proteins, including several involved in CME (16). Intersectin has two N-terminal Eps15 homology domains that are responsible for binding to epsin, SCAMP1, and numb (17-19), a central coil-coiled domain that interacts with Eps15 and SNAP-23 and -25 (17, 20, 21), and five SH3 domains in its C-terminal region that interact with multiple proline-rich domain proteins, including synaptojanin, dynamin, N-WASP, CdGAP, and mSOS (16, 22-25). The rich binding capability of intersectin has linked it to various functions from CME (17, 26, 27) and signaling (22, 28, 29) to mitogenesis (30, 31) and regulation of the actin cytoskeleton (23).Intersectin functions in SV recycling at the neuromuscular junction of Drosophila and C. elegans where it acts as a scaffold, regulating the synaptic levels of endocytic accessory proteins (21, 32-34). In vertebrates, the intersectin gene is subject to alternative splicing, and a longer isoform (intersectin-l) is generated that is expressed exclusively in neurons (26, 28, 35, 36). This isoform has all the binding modules of its short (intersectin-s) counterpart but also has additional domains: a DH and a PH domain that provide guanine nucleotide exchange factor (GEF) activity specific for Cdc42 (23, 37) and a C2 domain at the C terminus. Through its GEF activity and binding to actin regulatory proteins, including N-WASP, intersectin-l has been implicated in actin regulation and the development of dendritic spines (19, 23, 24). In addition, because the rest of the binding modules are shared between intersectin-s and -l, it is generally thought that the two intersectin isoforms have the same endocytic functions. In particular, given the well defined role for the invertebrate orthologs of intersectin-s in SV endocytosis, it is thought that intersectin-l performs this role in mammalian neurons, which lack intersectin-s. Defining the complement of intersectin functional activities in mammalian neurons is particularly relevant given that the protein is involved in the pathophysiology of Down syndrome (DS). Specifically, the intersectin gene is localized on chromosome 21q22.2 and is overexpressed in DS brains (38). Interestingly, alterations in endosomal pathways are a hallmark of DS neurons and neurons from the partial trisomy 16 mouse, Ts65Dn, a model for DS (39, 40). Thus, an endocytic trafficking defect may contribute to the DS disease process.Here, the functional roles of intersectin-l were studied in cultured hippocampal neurons. We find that intersectin-l is localized to the somatodendritic regions of neurons, where it co-localizes with CHC and AP-2 and regulates the uptake of transferrin. Intersectin-l also co-localizes with actin at dendritic spines and disrupting intersectin-l function alters dendritic spine development. In contrast, intersectin-l is absent from presynaptic terminals and has little or no role in SV recycling.     

Sortase D Forms the Covalent Bond That Links BcpB to the Tip of Bacillus cereus Pili

Bacillus cereus and other Gram-positive bacteria elaborate pili via a sortase D-catalyzed transpeptidation mechanism from major and minor pilin precursor substrates. After cleavage of the LPXTG sorting signal of the major pilin, BcpA, sortase D forms an amide bond between the C-terminal threonine and the amino group of lysine within the YPKN motif of another BcpA subunit. Pilus assembly terminates upon sortase A cleavage of the BcpA sorting signal, resulting in a covalent bond between BcpA and the cell wall cross-bridge. Here, we show that the IPNTG sorting signal of BcpB, the minor pilin, is cleaved by sortase D but not by sortase A. The C-terminal threonine of BcpB is amide-linked to the YPKN motif of BcpA, thereby positioning BcpB at the tip of pili. Thus, unique attributes of the sorting signals of minor pilins provide Gram-positive bacteria with a universal mechanism ordering assembly of pili.Sortases catalyze transpeptidation reactions to assemble proteins in the envelope of Gram-positive bacteria (1). Secreted proteins require a C-terminal sorting signal for sortase recognition such that sortase cleaves the substrate at a short peptide motif and forms a thioester-linked intermediate to its active site cysteine (2–4). Nucleophilic attack by an amino group within the bacterial envelope resolves the thioester intermediate, generating an amide bond tethering surface proteins at their C terminus onto Gram-positive bacteria (5). Four classes of sortases can be distinguished on the basis of sequence homology and substrate recognition (6, 7). Sortase A cleaves secreted protein at LPXTG sorting signals and recognizes the amino group of lipid II peptidoglycan precursors as a nucleophile (8, 9). Sortase B cleaves protein substrates at NPQTN sorting signals (10). This enzyme immobilizes proteins within fully assembled cell walls, utilizing the cell wall cross-bridge as a nucleophile (11). Sortase C cuts LPNTA sorting signals and anchors proteins to the peptidoglycan cross-bridges in sporulating bacteria (12, 13). Finally, sortase D catalyzes transpeptidation reactions in the assembly of pili (14, 15). Sortase D recognizes the amino group of lysine residues within the YPKN motif of pilin subunits as nucleophiles (16). The resultant sortase D-catalyzed amide bond links adjacent pilin subunits to grow the pilus fiber (16, 17).Pili of Gram-positive bacteria comprised either two or three different pilin subunits synthesized as cytoplasmic precursors with N-terminal signal peptides and C-terminal sorting signals (P1 precursors) (14, 18). After translocation across the plasma membrane, P2 precursor species arise from removal of the signal peptide from P1 precursors by a signal peptidase (16). Bacillus cereus pili are composed of two subunits; that is, the major pilin, BcpA, and the minor pilin, BcpB (15). In contrast to BcpA, which is deposited throughout the pilus, BcpB is found at fiber tip (15). Sortase D cleaves the BcpA LPXTG motif sorting signal between the threonine and glycine residues to form an amide bond to the ε-amino group of the lysine within the YPKN motif of adjacent BcpA subunits (16). However, sortase A also cleaves BcpA precursors, which are subsequently linked to the side chain amino group of meso-diaminopimelic acid within lipid II (19). The latter reaction serves to terminate fiber elongation, immobilizing BcpA pili in the cell wall envelope (19).The conservation of sortase D, the YPKN motif, and C-terminal sorting signal in major pilin subunits suggest a universal pilus assembly mechanism among Gram-positive bacteria (14, 20). However, the molecular mechanism whereby bacilli deposit BcpB, the minor pilin, at the tip of BcpA pili is not known. Although the BcpB precursor harbors an N-terminal signal peptide and a C-terminal IPNTG sorting signal, it lacks the YPKN pilin motif of the major subunit (15). Furthermore, the substrate properties of the BcpB IPNTG sorting signal for the four classes of sortases expressed by bacilli has yet to be established.     

Generation of Cubic Membranes by Controlled Homotypic Interaction of Membrane Proteins in the Endoplasmic Reticulum

Cell membranes predominantly consist of lamellar lipid bilayers. When studied in vitro, however, many membrane lipids can exhibit non-lamellar morphologies, often with cubic symmetries. An open issue is how lipid polymorphisms influence organelle and cell shape. Here, we used controlled dimerization of artificial membrane proteins in mammalian tissue culture cells to induce an expansion of the endoplasmic reticulum (ER) with cubic symmetry. Although this observation emphasizes ER architectural plasticity, we found that the changed ER membrane became sequestered into large autophagic vacuoles, positive for the autophagy protein LC3. Autophagy may be targeting irregular membrane shapes and/or aggregated protein. We suggest that membrane morphology can be controlled in cells.The observation that simple mixtures of amphiphilic (polar) lipids and water yield a rich flora of phase structures has opened a long-standing debate as to whether such membrane polymorphisms are relevant for living organisms (1–7). Lipid bilayers with planar geometry, termed lamellar symmetry, dominate the membrane structure of cells. However, this architecture comprises only a fraction of the structures seen with in vitro lipid-water systems (7–11). The propensity to form lamellar bilayers (a property exclusive to cylindrically shaped lipids) is flanked by a continuum of lipid structures that occur in a number of exotic and probably non-physiological non-bilayer configurations (3, 12). However, certain lipids, particularly those with smaller head groups and more bulky hydrocarbon chains, can adopt bilayered non-lamellar phases called cubic phases. Here the bilayer is curved everywhere in the form of saddle shapes corresponding to an energetically favorable minimal surface of zero mean curvature (1, 7). Because a substantial number of the lipids present in biological membranes, when studied as individual pure lipids, form cubic phases (13), cubic membranes have received particular interest in cell biology.Since the application of electron microscopy (EM)³ to the study of cell ultrastructure, unusual membrane morphologies have been reported for virtually every organelle (14, 15). However, interpretation of three-dimensional structures from two-dimensional electron micrographs is not easy (16). In seminal work, Landh (17) developed the method of direct template correlative matching, a technique that unequivocally assesses the presence of cubic membranes in biological specimens (16). Cubic phases adopt mathematically well defined three-dimensional configurations whose two-dimensional analogs have been derived (4, 17). In direct template correlative matching, electron micrographs are matched to these analogs. Cubic cell membrane geometries and in vitro cubic phases of purified lipid mixtures do differ in their lattice parameters; however, such deviations are thought to relate to differences in water activity and lipid to protein ratios (10, 14, 18). Direct template correlative matching has revealed thousands of examples of cellular cubic membranes in a broad survey of electron micrographs ranging from protozoa to human cells (14, 17) and, more recently, in the mitochondria of amoeba (19) and in subcellular membrane compartments associated with severe acute respiratory syndrome virus (20). Analysis of cellular cubic membranes has also been furthered by the development of EM tomography that confirmed the presence of cubic bilayers in the mitochondrial membranes of amoeba (21, 22).Although it is now clear that cubic membranes can exist in living cells, the generation of such architecture would appear tightly regulated, as evidenced by the dominance of lamellar bilayers in biology. In this light, we examined the capability and implications of generating cubic membranes in the endoplasmic reticulum (ER) of mammalian tissue culture cells. The ER is a spatially interconnected complex consisting of two domains, the nuclear envelope and the peripheral ER (23–26). The nuclear envelope surrounds the nucleus and is composed of two continuous sheets of membranes, an inner and outer nuclear membrane connected to each other at nuclear pores. The peripheral ER constitutes a network of branching trijunctional tubules that are continuous with membrane sheet regions that occur in closer proximity to the nucleus. Recently it has been suggested that the classical morphological definition of rough ER (ribosome-studded) and smooth ER (ribosome-free) may correspond to sheet-like and tubular ER domains, respectively (27). The ER has a strong potential for cubic architectures, as demonstrated by the fact that the majority of cubic cell membranes in the EM record come from ER-derived structures (14, 17). Furthermore, ER cubic symmetries are an inducible class of organized smooth ER (OSER), a definition collectively referring to ordered smooth ER membranes (=stacked cisternae on the outer nuclear membrane, also called Karmelle (28–30), packed sinusoidal ER (31), concentric membrane whorls (30, 32–34), and arrays of crystalloid ER (35–37)). Specifically, weak homotypic interactions between membrane proteins produce both a whorled and a sinusoidal OSER phenotype (38), the latter exhibiting a cubic symmetry (16, 39).We were able to produce OSER with cubic membrane morphology via induction of homo-dimerization of artificial membrane proteins. Interestingly, the resultant cubic membrane architecture was removed from the ER system by incorporation into large autophagic vacuoles. To assess whether these cubic symmetries were favored in the absence of cellular energy, we depleted ATP. To our surprise, the cells responded by forming large domains of tubulated membrane, suggesting that a cubic symmetry was not the preferred conformation of the system. Our results suggest that whereas the endoplasmic reticulum is capable of adopting cubic symmetries, both the inherent properties of the ER system and active cellular mechanisms, such as autophagy, can tightly control their appearance.     

Secretory Carrier Membrane Protein 2 Regulates Cell-surface Targeting of Brain-enriched Na+/H+ Exchanger NHE5

NHE5 is a brain-enriched Na⁺/H⁺ exchanger that dynamically shuttles between the plasma membrane and recycling endosomes, serving as a mechanism that acutely controls the local pH environment. In the current study we show that secretory carrier membrane proteins (SCAMPs), a group of tetraspanning integral membrane proteins that reside in multiple secretory and endocytic organelles, bind to NHE5 and co-localize predominantly in the recycling endosomes. In vitro protein-protein interaction assays revealed that NHE5 directly binds to the N- and C-terminal cytosolic extensions of SCAMP2. Heterologous expression of SCAMP2 but not SCAMP5 increased cell-surface abundance as well as transporter activity of NHE5 across the plasma membrane. Expression of a deletion mutant lacking the SCAMP2-specific N-terminal cytosolic domain, and a mini-gene encoding the N-terminal extension, reduced the transporter activity. Although both Arf6 and Rab11 positively regulate NHE5 cell-surface targeting and NHE5 activity across the plasma membrane, SCAMP2-mediated surface targeting of NHE5 was reversed by dominant-negative Arf6 but not by dominant-negative Rab11. Together, these results suggest that SCAMP2 regulates NHE5 transit through recycling endosomes and promotes its surface targeting in an Arf6-dependent manner.Neurons and glial cells in the central and peripheral nervous systems are especially sensitive to perturbations of pH (1). Many voltage- and ligand-gated ion channels that control membrane excitability are sensitive to changes in cellular pH (1-3). Neurotransmitter release and uptake are also influenced by cellular and organellar pH (4, 5). Moreover, the intra- and extracellular pH of both neurons and glia are modulated in a highly transient and localized manner by neuronal activity (6, 7). Thus, neurons and glia require sophisticated mechanisms to finely tune ion and pH homeostasis to maintain their normal functions.Na⁺/H⁺ exchangers (NHEs)³ were originally identified as a class of plasma membrane-bound ion transporters that exchange extracellular Na⁺ for intracellular H⁺, and thereby regulate cellular pH and volume. Since the discovery of NHE1 as the first mammalian NHE (8), eight additional isoforms (NHE2-9) that share 25-70% amino acid identity have been isolated in mammals (9, 10). NHE1-5 commonly exhibit transporter activity across the plasma membrane, whereas NHE6-9 are mostly found in organelle membranes and are believed to regulate organellar pH in most cell types at steady state (11). More recently, NHE10 was identified in human and mouse osteoclasts (12, 13). However, the cDNA encoding NHE10 shares only a low degree of sequence similarity with other known members of the NHE gene family, raising the possibility that this sodium-proton exchanger may belong to a separate gene family distantly related to NHE1-9 (see Ref. 9).NHE gene family members contain 12 putative transmembrane domains at the N terminus followed by a C-terminal cytosolic extension that plays a role in regulation of the transporter activity by protein-protein interactions and phosphorylation. NHEs have been shown to regulate the pH environment of synaptic nerve terminals and to regulate the release of neurotransmitters from multiple neuronal populations (14-16). The importance of NHEs in brain function is further exemplified by the findings that spontaneous or directed mutations of the ubiquitously expressed NHE1 gene lead to the progression of epileptic seizures, ataxia, and increased mortality in mice (17, 18). The progression of the disease phenotype is associated with loss of specific neuron populations and increased neuronal excitability. However, NHE1-null mice appear to develop normally until 2 weeks after birth when symptoms begin to appear. Therefore, other mechanisms may compensate for the loss of NHE1 during early development and play a protective role in the surviving neurons after the onset of the disease phenotype.NHE5 was identified as a unique member of the NHE gene family whose mRNA is expressed almost exclusively in the brain (19, 20), although more recent studies have suggested that NHE5 might be functional in other cell types such as sperm (21, 22) and osteosarcoma cells (23). Curiously, mutations found in several forms of congenital neurological disorders such as spinocerebellar ataxia type 4 (24-26) and autosomal dominant cerebellar ataxia (27-29) have been mapped to chromosome 16q22.1, a region containing NHE5. However, much remains unknown as to the molecular regulation of NHE5 and its role in brain function.Very few if any proteins work in isolation. Therefore identification and characterization of binding proteins often reveal novel functions and regulation mechanisms of the protein of interest. To begin to elucidate the biological role of NHE5, we have started to explore NHE5-binding proteins. Previously, β-arrestins, multifunctional scaffold proteins that play a key role in desensitization of G-protein-coupled receptors, were shown to directly bind to NHE5 and promote its endocytosis (30). This study demonstrated that NHE5 trafficking between endosomes and the plasma membrane is regulated by protein-protein interactions with scaffold proteins. More recently, we demonstrated that receptor for activated C-kinase 1 (RACK1), a scaffold protein that links signaling molecules such as activated protein kinase C, integrins, and Src kinase (31), directly interacts with and activates NHE5 via integrin-dependent and independent pathways (32). These results further indicate that NHE5 is partly associated with focal adhesions and that its targeting to the specialized microdomain of the plasma membrane may be regulated by various signaling pathways.Secretory carrier membrane proteins (SCAMPs) are a family of evolutionarily conserved tetra-spanning integral membrane proteins. SCAMPs are found in multiple organelles such as the Golgi apparatus, trans-Golgi network, recycling endosomes, synaptic vesicles, and the plasma membrane (33, 34) and have been shown to play a role in exocytosis (35-38) and endocytosis (39). Currently, five isoforms of SCAMP have been identified in mammals. The extended N terminus of SCAMP1-3 contain multiple Asn-Pro-Phe (NPF) repeats, which may allow these isoforms to participate in clathrin coat assembly and vesicle budding by binding to Eps15 homology (EH)-domain proteins (40, 41). Further, SCAMP2 was shown recently to bind to the small GTPase Arf6 (38), which is believed to participate in traffic between the recycling endosomes and the cell surface (42, 43). More recent studies have suggested that SCAMPs bind to organellar membrane type NHE7 (44) and the serotonin transporter SERT (45) and facilitate targeting of these integral membrane proteins to specific intracellular compartments. We show in the current study that SCAMP2 binds to NHE5, facilitates the cell-surface targeting of NHE5, and elevates Na⁺/H⁺ exchange activity at the plasma membrane, whereas expression of a SCAMP2 deletion mutant lacking the N-terminal domain containing the NPF repeats suppresses the effect. Further we show that this activity of SCAMP2 requires an active form of a small GTPase Arf6, but not Rab11. We propose a model in which SCAMPs bind to NHE5 in the endosomal compartment and control its cell-surface abundance via an Arf6-dependent pathway.     

Adenine Nucleotide-dependent Regulation of Assembly of Bacterial Tubulin-like FtsZ by a Hypermorph of Bacterial Actin-like FtsA

Cytokinesis in bacteria depends upon the contractile Z ring, which is composed of dynamic polymers of the tubulin homolog FtsZ as well as other membrane-associated proteins such as FtsA, a homolog of actin that is required for membrane attachment of the Z ring and its subsequent constriction. Here we show that a previously characterized hypermorphic mutant FtsA (FtsA*) partially disassembled FtsZ polymers in vitro. This effect was strictly dependent on ATP or ADP binding to FtsA* and occurred at substoichiometric levels relative to FtsZ, similar to cellular levels. Nucleotide-bound FtsA* did not affect FtsZ GTPase activity or the critical concentration for FtsZ assembly but was able to disassemble preformed FtsZ polymers, suggesting that FtsA* acts on FtsZ polymers. Microscopic examination of the inhibited FtsZ polymers revealed a transition from long, straight polymers and polymer bundles to mainly short, curved protofilaments. These results indicate that a bacterial actin, when activated by adenine nucleotides, can modify the length distribution of bacterial tubulin polymers, analogous to the effects of actin-depolymerizing factor/cofilin on F-actin.Bacterial cell division requires a large number of proteins that colocalize to form a putative protein machine at the cell membrane (1). This machine, sometimes called the divisome, recruits enzymes to synthesize the septum cell wall and to initiate and coordinate the invagination of the cytoplasmic membrane (and in Gram-negative bacteria, the outer membrane). The most widely conserved and key protein for this process is FtsZ, a homolog of tubulin that forms a ring structure called the Z ring, which marks the site of septum formation (2, 3). Like tubulin, FtsZ assembles into filaments with GTP but does not form microtubules (4). The precise assembly state and conformation of these FtsZ filaments at the division ring is not clear, although recent electron tomography work suggests that the FtsZ ring consists of multiple short filaments tethered to the membrane at discrete junctures (5), which may represent points along the filaments bridged by membrane anchor proteins.In Escherichia coli, two of these anchor proteins are known. One of these, ZipA, is not well conserved but is an essential protein in E. coli. ZipA binds to the C-terminal tail of FtsZ (6–8), and purified ZipA promotes bundling of FtsZ filaments in vitro (9, 10). The other, FtsA, is also essential in E. coli and is more widely conserved among bacterial species. FtsA is a member of the HSP70/actin superfamily (11, 12), and like ZipA, it interacts with the C-terminal tail of FtsZ (7, 13–15). FtsA can self-associate (16, 17) and bind ATP (12, 18), but reports of ATPase activity vary, with Bacillus subtilis FtsA having high activity (19) and Streptococcus pneumoniae FtsA exhibiting no detectable activity (20). There are no reports of any other in vitro activities of FtsA, including effects on FtsZ assembly.Understanding how FtsA affects FtsZ assembly is important because FtsA has a number of key activities in the cell. It is required for recruitment of a number of divisome proteins (21, 22) and helps to tether the Z ring to the membrane via a C-terminal membrane-targeting sequence (23). FtsA, like ZipA and other divisome proteins, is necessary to activate the contraction of the Z ring (24, 25). In E. coli, the FtsA:FtsZ ratio is crucial for proper cell division, with either too high or too low a ratio inhibiting septum formation (26, 27). This ratio is roughly 1:5, with ∼700 molecules of FtsA and 3200 molecules of FtsZ per cell (28), which works out to concentrations of 1–2 and 5–10 μm, respectively.Another interesting property of FtsA is that single residue alterations in the protein can result in significant enhancement of divisome activity. For example, the R286W mutation of FtsA, also called FtsA*, can substitute for the native FtsA and divide the cell. However, this mutant FtsA causes E. coli cells to divide at less than 80% of their normal length (29) and allows efficient division of E. coli cells in the absence of ZipA (30), indicating that it has gain-of-function activity. FtsA* and other hypermorphic mutations such as E124A and I143L can also increase division activity in cells lacking other essential divisome components (31–33). The R286W and E124A mutants of FtsA also bypass the FtsA:FtsZ ratio rule, allowing cell division to occur at higher ratios than with WT² FtsA. This may be because the altered FtsA proteins self-associate more readily than WT FtsA, which may cause different changes in FtsZ assembly state as compared with WT FtsA (17, 34).In this study, we use an in vitro system with purified FtsZ and a purified tagged version of FtsA* to elucidate the role of FtsA in activating constriction of the Z ring in vivo. We show that FtsA*, at physiological concentrations in the presence of ATP or ADP, has significant effects on the assembly of FtsZ filaments.