A remote CheZ orthologue retains phosphatase function

Aspartyl‐phosphate phosphatases underlie the rapid responses of bacterial chemotaxis. One such phosphatase, CheZ, was originally proposed to be restricted to beta and gamma proteobacter, suggesting only a small subset of microbes relied on this protein. A putative CheZ phosphatase was identified genetically in the epsilon proteobacter Helicobacter pylori (Mol Micro 61:187). H. pylori utilizes a chemotaxis system consisting of CheAY, three CheVs, CheW, CheY_HP and the putative CheZ to colonize the host stomach. Here we investigate whether this CheZ has phosphatase activity. We phosphorylated potential targets in vitro using either a phosphodonor or the CheAY kinase and [γ‐³²P]‐ATP, and found that H. pylori CheZ (CheZ_HP) efficiently dephosphorylates CheY_HP and CheAY and has additional weak activity on CheV2. We detected no phosphatase activity towards CheV1 or CheV3. Mutations corresponding to Escherichia coli CheZ active site residues or deletion of the C‐terminal region inactivate CheZ_HP phosphatase activity, suggesting the two CheZs function similarly. Bioinformatics analysis suggests that CheZ phosphatases are found in all proteobacteria classes, as well as classes Aquificae, Deferribacteres, Nitrospira and Sphingobacteria, demonstrating that CheZ phosphatases are broadly distributed within Gram‐negative bacteria.     

细菌趋化信号通路中的磷酸酯酶CheZ

细菌通过趋化系统获得在复杂环境中的生存优势。趋化性在细菌致病性、病菌定殖、固氮细菌与宿主共生、植物与微生物互作等方面有重要的作用。作为趋化信号适应中不可或缺的调节蛋白,对CheZ的深入研究具有重要意义。本文主要对CheZ的结构、作用机制、功能调节、蛋白定位以及进化地位等方面的研究现状进行了综述,旨在为其它细菌中趋化系统的研究提供有益参考。     

Structure and catalytic mechanism of the E. coli chemotaxis phosphatase CheZ

The protein CheZ, which has the last unknown structure in the Escherichia coli chemotaxis pathway, stimulates the dephosphorylation of the response regulator CheY by an unknown mechanism. Here we report the co-crystal structure of CheZ with CheY, Mg(2+) and the phosphoryl analog, BeF(3)(-). The predominant structural feature of the CheZ dimer is a long four-helix bundle composed of two helices from each monomer. The side chain of Gln 147 of CheZ inserts into the CheY active site and is essential to the dephosphorylation activity of CheZ. Gln 147 may orient a water molecule for nucleophilic attack, similar to the role of the conserved Gln residue in the RAS family of GTPases. Similarities between the CheY[bond] CheZ and Spo0F [bond]Spo0B structures suggest a general mode of interaction for modulation of response regulator phosphorylation chemistry.     

The nontransmembrane tyrosine phosphatase PTP-1B localizes to the endoplasmic reticulum via its 35 amino acid C-terminal sequence.

We report the first intracellular characterization of an endogenous nontransmembrane protein tyrosine phosphatase (PTP). Using affinity-purified polyclonal antibodies, we have identified PTP-1B as a 50 kd serine phosphoprotein in immunoprecipitation and immunoblotting assays. Surprisingly, indirect immunofluorescence experiments indicate that PTP-1B is localized predominantly in the endoplasmic reticulum (ER). Subcellular fractionation is consistent with this localization and establishes that PTP-1B is tightly associated with microsomal membranes, with its phosphatase domain oriented towards the cytoplasm. The C-terminal 35 amino acids of PTP-1B are both necessary and sufficient for targeting to the ER. The finding of a tyrosine phosphatase on the ER suggests new possibilities for cellular events controlled by tyrosine phosphorylation.     

The accessibility of cys-120 in CheA(S) is important for the binding of CheZ and enhancement of CheZ phosphatase activity

The cheA gene of Escherichia coli encodes two proteins from in-frame tandem translation start sites. The long form of CheA (CheA(L)) is the histidine kinase responsible for phosphorylating the response regulator, CheY. The short form of CheA (CheA(S)) is identical in domain structure to CheA(L) except that it is missing the first 97 amino acids. Reduced CheA(S) bound to and enhanced the activity of the phosphatase of phospho-CheY, CheZ. Oxidized CheA(S) was unable to interact with CheZ. Oxidized CheA(S) formed covalent dimers, whereas CheA(L) did not. This property was believed to be the result of an intermolecular disulfide bond. The CheA proteins contain three cysteine residues, one of which likely lies within the CheZ binding region of CheA(S) and is exposed to solvent. We identified the CheZ binding domain of CheA(S) by testing the various fragments of CheA(S) that contain cysteine residues for CheZ binding activity in an ELISA-based CheA(S)-CheZ binding assay. Fragments of CheA(S) lacking the truncated P1 domain of CheA(S) ('P1) were unable to bind CheZ. We also found that a fusion of the first 42 amino acids of CheA(S) ('P1 domain) to GST bound CheZ and enhanced its activity. The interaction between the GST-CheA[98-139] fusion protein and CheZ was dependent on the accessibility of a cysteine residue (Cys-120) located in the 'P1 domain.     

Mutations in the chemotactic response regulator, CheY, that confer resistance to the phosphatase activity of CheZ

CheY, a small cytoplasmic response regulator, plays an essential role in the chemotaxis pathway. The concentration of phospho-CheY is thought to determine the swimming behaviour of the cell: high levels of phospho-CheY cause bacteria to rotate their flagella clockwise and tumble, whereas low levels of the phos-phorylated form of the protein allow counter-ciockwise rotation of the flagella and smooth swimming. The phosphorylation state of CheY in vivo is determined by the activity of the phosphoryl donor CheA, and by the antagonistic effect of dephosphorylation of phospho-CheY. The dephosphorylation rate is controlled by the intrinsic autohydrolytic activity of phospho-CheY and by the CheZ protein, which accelerates dephosphorylation. We have analysed the effect of CheZ on the dephosphorylation rates of several mutant CheY proteins. Two point mutations were identified which were 50-fold and 5-fold less sensitive to the activity of CheZ than was the wild-type protein. Nonetheless, the phosphorylation and autodephos-phorylation rates of these mutants, CheY23ND and CheY26KE, were observed to be identical to those of wild-type CheY in the absence of CheZ. These are the first examples of CheY mutations that reduce sensitivity to the phosphatase activity of CheZ without being altered in terms of their intrinsic phosphorylation and autodephospborylation rates, interestingly, the residues Asn-23 and Lys-26 are located on a face of CheY far from the phosphorylation site (Asp-57), distinct from the previously described site of inter-action with the histidine kinase CheA, and partially overlapping with a region implicated in interaction with the flagellar switch.     

Kinetic characterization of catalysis by the chemotaxis phosphatase CheZ. Modulation of activity by the phosphorylated CheY substrate

CheZ catalyzes the dephosphorylation of the response regulator CheY in the two-component regulatory system that mediates chemotaxis in Escherichia coli. CheZ is a homodimer with two active sites for dephosphorylation. To gain insight into cellular mechanisms for the precise regulation of intracellular phosphorylated CheY (CheYp) levels, we evaluated the kinetic properties of CheZ. The steady state rate of CheZ-mediated dephosphorylation of CheYp displayed marked sigmoidicity with respect to CheYp concentration and a k(cat) of 4.9 s(-1). In contrast, the gain of function mutant CheZ-I21T with an amino acid substitution far from the active site gave hyperbolic kinetics and required far lower CheYp for half-saturation but had a similar k(cat) value as the wild type enzyme. Stopped flow fluorescence measurements demonstrated a 6-fold faster CheZ/CheYp association rate for CheZ-I21T (k(assoc) = 3.4 x 10(7) M (-1) s(-1)) relative to wild type CheZ (k(assoc) = 5.6 x 10(6) M(-1) s(-1)). Dissociation of the CheZ.CheYBeF(3) complex was slow for both wild type CheZ (k(dissoc) = 0.040 s(-1)) and CheZ-I21T (k(dissoc) = 0.023 s(-1)) and, when taken with the k(assoc) values, implied K(d) values of 7.1 and 0.68 nm, respectively. However, comparison of the k(dissoc) and k(cat) values implied that CheZ and CheYp are not at binding equilibrium during catalysis and that once CheYp binds, it is almost always dephosphorylated. The rate constants were collated to formulate a kinetic model for CheZ-mediated dephosphorylation that includes autoregulation by CheYp and allowed prediction of CheZ activities at CheZ and CheYp concentrations likely to be present in cells.     

Stabilization of polar localization of a chemoreceptor via its covalent modifications and its communication with a different chemoreceptor

In the chemotaxis of Escherichia coli, polar clustering of the chemoreceptors, the histidine kinase CheA, and the adaptor protein CheW is thought to be involved in signal amplification and adaptation. However, the mechanism that leads to the polar localization of the receptor is still largely unknown. In this study, we examined the effect of receptor covalent modification on the polar localization of the aspartate chemoreceptor Tar fused to green fluorescent protein (GFP). Amidation (and presumably methylation) of Tar-GFP enhanced its own polar localization, although the effect was small. The slight but significant effect of amidation on receptor localization was reinforced by the fact that localization of a noncatalytic mutant version of GFP-CheR that targets to the C-terminal pentapeptide sequence of Tar was similarly facilitated by receptor amidation. Polar localization of the demethylated version of Tar-GFP was also enhanced by increasing levels of the serine chemoreceptor Tsr. The effect of covalent modification on receptor localization by itself may be too small to account for chemotactic adaptation, but receptor modification is suggested to contribute to the molecular assembly of the chemoreceptor/histidine kinase array at a cell pole, presumably by stabilizing the receptor dimer-to-dimer interaction.     

High mobility of carboxyl-terminal region of bacterial chemotaxis phosphatase CheZ is diminished upon binding divalent cation or CheY-P substrate

In Escherichia coli chemotaxis, the CheZ phosphatase catalyzes the removal of the phosphoryl group from the signaling molecule, CheY. The cocrystal structure of CheZ with CheY x BeF3- x Mg2+ (a stable analogue of CheY-P) revealed that CheZ is a homodimer with a multidomain, nonglobular structure. To explore the effects of CheZ/CheY complex formation on CheZ structure, the rotational dynamics of the different structural domains of CheZ [the four-helix bundle, the N-terminal helix, the C-terminal helix, and the putative disordered linker between the C-terminal helix and the bundle] were evaluated. To monitor dynamics of the different regions, fluorescein probes were covalently attached at various locations on CheZ through reaction with engineered cysteine residues and the rotational behavior of the fluoresceinated derivatives were assessed using steady state fluorescence anisotropy. Anisotropy measurements at various solution viscosities (Perrin plot analysis) demonstrated large differences in global rotational motion for fluorophores located on different regions. Rotational correlation times for probes located on the four-helix bundle and the N-terminal helix agreed well with theoretical values predicted for a protein the size and shape of the four-helix bundle. However, the rotational correlation times of probes located on the linker and the C-terminal helix were 8-20x lower, indicating rapid motion independent of the bundle. The anisotropies of probes located on the linker and the C-terminal helix increased in the presence of divalent cation (Mg2+, Ca2+, or Mn2+) in a saturable fashion, consistent with a binding event (Kd approximately 1-4 mM) that results in decreased mobility. The anisotropies of probes located on the C-terminal helix and the C-terminal portion of the linker increased further as a result of binding CheY-P. In light of the recently available structural data and the high independent mobility of the C-terminus demonstrated here, we interpret the CheY-P-dependent increase in anisotropy to be a consequence of decreased mobility of the C-terminal region due to binding interactions with CheY-P, and not to the formation of higher order aggregates of the CheZ2(CheY-P)2 complex.     

The C. elegans RSA complex localizes protein phosphatase 2A to centrosomes and regulates mitotic spindle assembly

Microtubule behavior changes during the cell cycle and during spindle assembly. However, it remains unclear how these changes are regulated and coordinated. We describe a complex that targets the Protein Phosphatase 2A holoenzyme (PP2A) to centrosomes in C. elegans embryos. This complex includes Regulator of Spindle Assembly 1 (RSA-1), a targeting subunit for PP2A, and RSA-2, a protein that binds and recruits RSA-1 to centrosomes. In contrast to the multiple functions of the PP2A catalytic subunit, RSA-1 and RSA-2 are specifically required for microtubule outgrowth from centrosomes and for spindle assembly. The centrosomally localized RSA-PP2A complex mediates these functions in part by regulating two critical mitotic effectors: the microtubule destabilizer KLP-7 and the C. elegans regulator of spindle assembly TPXL-1. By regulating a subset of PP2A functions at the centrosome, the RSA complex could therefore provide a means of coordinating microtubule outgrowth from centrosomes and kinetochore microtubule stability during mitotic spindle assembly.     

Lysosomal acid phosphatase is transported via endosomes to lysosomes.

Involvement of endosomes in transport of newly synthesized acid phosphatase to lysosomes was investigated using the Golgi fraction (GF1 + 2), enriched in endosomes. The Golgi fraction (GF1 + 2) was prepared from the livers of rats given [35S]methionine and asialofetuin conjugated-horseradish peroxidase (HRP). Newly synthesized acid phosphatase in the endosomes containing internalized asialofetuin-HRP was measured as a loss of the detectable labeled enzyme after 3,3'-diaminobenzidine (DAB) and H2O2 reaction, due to formation of insoluble polymers which reduce protein antigenicity. With this procedure, acid phosphatase was all but undetectable in the Golgi fraction. Thus, newly synthesized acid phosphatase is apparently transported to lysosomes by endosomes.     

The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis

PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics.     

PLRP2 selectively localizes synaptic membrane proteins via acyl-chain remodeling of phospholipids

The plasma membrane of neurons consists of distinct domains, each of which carries specialized functions and a characteristic set of membrane proteins. While this compartmentalized membrane organization is essential for neuronal functions, it remains controversial how neurons establish these domains on the laterally fluid membrane. Here, using immunostaining, lipid-MS analysis and gene ablation with the CRISPR/Cas9 system, we report that the pancreatic lipase-related protein 2 (PLRP2), a phospholipase A1 (PLA1), is a key organizer of membrane protein localization at the neurite tips of PC12 cells. PLRP2 produced local distribution of 1-oleoyl-2-palmitoyl-PC at these sites through acyl-chain remodeling of membrane phospholipids. The resulting lipid domain assembled the syntaxin 4 (Stx4) protein within itself by selectively interacting with the transmembrane domain of Stx4. The localized Stx4, in turn, facilitated the fusion of transport vesicles that contained the dopamine transporter with the domain of the plasma membrane, which led to the localized distribution of the transporter to that domain. These results revealed the pivotal roles of PLA1, specifically PLRP2, in the formation of functional domains in the plasma membrane of neurons. In addition, our results suggest a mode of membrane organization in which the local acyl-chain remodeling of membrane phospholipids controls the selective localization of membrane proteins by regulating both lipid-protein interactions and the fusion of transport vesicles to the lipid domain.     

ATP-binding cassette transporter isoform C2 localizes to the apical plasma membrane via interactions with scaffolding protein

ATP-binding cassette transporter isoform C2 (ABCC2) localizes to the apical plasma membrane in polarized cells. Apical localization of ABCC2 in hepatocytes plays an important role in biliary excretion of endobiotics and xenobiotics, but the mechanism by which ABCC2 localizes to the apical membrane has not been conclusively elucidated. Here, we investigate the role of scaffolding proteins on ABCC2 localization with a focus on the function of PDZK1 (post-synaptic density 95/disk large/zonula occludens-1 domain containing 1) in regulating ABCC2 localization. The C-terminal 77 residues of ABCC2 were used to probe interacting proteins from HepG2 cells. Protein mass fingerprinting identified PDZK1 as a major interacting protein. PDZK1 associated with the plasma membrane, most likely at the apical vacuoles of HepG2 cells. Affinity pull-down assays confirmed that the C-terminal NSTKF of ABCC2 bound to the fourth PDZ domain of PDZK1. Removal of this PDZ-binding motif significantly reduced the normal apical localization of ABCC2. In HepG2 cells, overexpression of this fourth domain overcame endogenous PDZK1 and reduced the ABCC2 localization at the apical membrane with a reciprocal increase of intracellular accumulation of mislocalized ABCC2. These results suggest a possible role for an interaction between ABCC2 and PDZK1 in apical localization of ABCC2 in hepatocytes.     

Macrophages survive hyperoxia via prolonged ERK activation due to phosphatase down-regulation

Macrophages exposed to hyperoxia in the lung continue to survive for prolonged periods. We previously reported (Nyunoya, T., Powers, L. S., Yarovinsky, T. O., Butler, N. S., Monick, M. M., and Hunninghake, G. W. (2003) J. Biol. Chem. 278, 36099-36106) that hyperoxia induces cell cycle arrest and sustained extracellular signal-related kinase (ERK) activity in macrophages. In this study, we determined the mechanisms of hyperoxia-induced ERK activation and how ERK activity plays a pro-survival role in hyperoxia-exposed cells. Inhibition of ERK activity decreased survival of hyperoxia-exposed macrophages. This was due, at least in part, to down-regulation of the pro-apoptotic Bcl-2 family member, BimEL. In determining the mechanism of ERK activation by hyperoxia, we found that ERK activation was not associated with hyperoxia-induced activation of the upstream ERK kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2. When we examined the ability of whole cell lysates from hyperoxia-exposed cells to dephosphorylate purified phosphorylated ERK, we found decreased ERK-directed phosphatase activity. Two particular ERK-directed phosphatases (protein phosphatase 2A and MAPK phosphatase-3) demonstrated decreased activity in hyperoxia-exposed cells. Moreover, whole cell lysates from normoxia-exposed cells depleted of PP2A or MAPK phosphatase-3 were also less able to dephosphorylate ERK. These data demonstrate that, in hyperoxia-exposed macrophages, sustained activation of ERK due to phosphatase down-regulation permits macrophage survival via effects on the balance between pro- and anti-apoptotic Bcl-2 family proteins.     

The archaeal exosome localizes to the membrane

We studied the cellular localization of the archaeal exosome, an RNA-processing protein complex containing orthologs of the eukaryotic proteins Rrp41, Rrp42, Rrp4 and Csl4, and an archaea-specific subunit annotated as DnaG. Fractionation of cell-free extracts of Sulfolobus solfataricus in sucrose density gradients revealed that DnaG and the active-site comprising subunit Rrp41 are enriched together with surface layer proteins in a yellow colored ring, implicating that the exosome is membrane-bound. In accordance with this assumption, DnaG and Rrp41 were detected at the periphery of the cell by immunofluorescence microscopy. Our finding suggests that RNA processing in Archaea is spatially organized.

Structured summary

MINT-7891213: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by cosedimentation in solution (MI:0028)MINT-7891235: Rrp41 (uniprotkb:Q9UXC2), DnaG (uniprotkb:P95980) and SlaA (uniprotkb:Q2M1E7) colocalize (MI:0403) by cosedimentation through density gradient (MI:0029)MINT-7891278: Rrp41 (uniprotkb:Q9UXC2) and DnaG (uniprotkb:P95980) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

Retromer localizes to autophagosomes during HCV replication

<正>Dear Editor,Hepatitis C virus(HCV)is a positive-strand RNA virus that belongs to the genus Hepacivirus within the Flaviviridae family.HCV causes chronic liver diseases,and185 million people are infected(Messina et al.,2015).Currently,there is no approved vaccine to prevent hepatitis C.HCV induces autophagy through elevating reactive oxygen species(ROS)levels via the unfolded