Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

2011 ◽  
Vol 441 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Gaëlle Tahay ◽  
Elsa Wiame ◽  
Donatienne Tyteca ◽  
Pierre J. Courtoy ◽  
Emile Van Schaftingen
Keyword(s):  
Catalytic Activity ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Kinetic Properties ◽  
Conserved Residues ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Membrane Region

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.

scholarly journals PKCε, Via its Regulatory Domain and Independently of its Catalytic Domain, Induces Neurite-like Processes in Neuroblastoma Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 713-726 ◽  
Author(s):  
Ruth Zeidman ◽  
Bjarne Löfgren ◽  
Sven Påhlman ◽  
Christer Larsson
Keyword(s):  
Neurite Outgrowth ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Neuroblastoma Cells ◽  
Dominant Negative ◽  
Regulatory Domain ◽  
Process Formation ◽  
Synaptic Markers ◽  
Green Fluorescent ◽  
Necessary And Sufficient

To investigate the role of protein kinase C (PKC) isoforms in regulation of neurite outgrowth, PKCα, βII, δ, and ε fused to enhanced green fluorescent protein (EGFP) were transiently overexpressed in neuroblastoma cells. Overexpression of PKCε–EGFP induced cell processes whereas the other isoforms did not. The effect of PKCε–EGFP was not suppressed by the PKC inhibitor GF109203X. Instead, process formation was more pronounced when the regulatory domain was introduced. Overexpression of various fragments from PKCε regulatory domain revealed that a region encompassing the pseudosubstrate, the two C1 domains, and parts of the V3 region were necessary and sufficient for induction of processes. By deleting the second C1 domain from this construct, a dominant-negative protein was generated which suppressed processes induced by full-length PKCε and neurites induced during retinoic acid- and growth factor–induced differentiation. As with neurites in differentiated neuroblastoma cells, processes induced by the PKCε– PSC1V3 protein contained α-tubulin, neurofilament-160, and F-actin, but the PKCε–PSC1V3-induced processes lacked the synaptic markers synaptophysin and neuropeptide Y.  These data suggest that PKCε, through its regulatory domain, can induce immature neurite-like processes via a mechanism that appears to be of importance for neurite outgrowth during neuronal differentiation.

scholarly journals The PWWP Domain of Dnmt3a and Dnmt3b Is Required for Directing DNA Methylation to the Major Satellite Repeats at Pericentric Heterochromatin

2004 ◽  
Vol 24 (20) ◽  
pp. 9048-9058 ◽  
Author(s):  
Taiping Chen ◽  
Naomi Tsujimoto ◽  
En Li
Keyword(s):  
Dna Methylation ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Regulatory Region ◽  
Pericentric Heterochromatin ◽  
Pwwp Domain ◽  
Major Satellite ◽  
Satellite Repeats ◽  
Associated Proteins ◽  
Green Fluorescent

ABSTRACT Dnmt3a and Dnmt3b are responsible for the establishment of DNA methylation patterns during development. These proteins contain, in addition to a C-terminal catalytic domain, a unique N-terminal regulatory region that harbors conserved domains, including a PWWP domain. The PWWP domain, characterized by the presence of a highly conserved proline-tryptophan-tryptophan-proline motif, is a module of 100 to 150 amino acids found in many chromatin-associated proteins. However, the function of the PWWP domain remains largely unknown. In this study, we provide evidence that the PWWP domains of Dnmt3a and Dnmt3b are involved in functional specialization of these enzymes. We show that both endogenous and green fluorescent protein-tagged Dnmt3a and Dnmt3b are particularly concentrated in pericentric heterochromatin. Mutagenesis analysis indicates that their PWWP domains are required for their association with pericentric heterochromatin. Disruption of the PWWP domain abolishes the ability of Dnmt3a and Dnmt3b to methylate the major satellite repeats at pericentric heterochromatin. Furthermore, we demonstrate that the Dnmt3a PWWP domain has little DNA-binding ability, in contrast to the Dnmt3b PWWP domain, which binds DNA nonspecifically. Collectively, our results suggest that the PWWP domains of Dnmt3a and Dnmt3b are essential for targeting these enzymes to pericentric heterochromatin, probably via a mechanism other than protein-DNA interactions.

scholarly journals Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 434
Author(s):  
Alison Mbekeani ◽  
Will Stanley ◽  
Vishal Kalel ◽  
Noa Dahan ◽  
Einat Zalckvar ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Metabolic Energy ◽  
Peroxisomal Protein ◽  
Genes Encoding ◽  
Peroxisomal Targeting ◽  
Membrane Bound ◽  
Green Fluorescent

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

scholarly journals Rice black-streaked dwarf virus P10 suppresses protein kinase C in insect vector through changing the subcellular localization of LsRACK1

2019 ◽  
Vol 374 (1767) ◽  
pp. 20180315 ◽  
Author(s):  
Lina Lu ◽  
Qi Wang ◽  
Deqing Huang ◽  
Qiufang Xu ◽  
Xueping Zhou ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Brown Planthopper ◽  
Subcellular Fractionation ◽  
Dwarf Virus ◽  
Insect Vector ◽  
Kinase C ◽  
Green Fluorescent

Rice black-streaked dwarf virus (RBSDV) was known to be transmitted by the small brown planthopper (SBPH) in a persistent, circulative and propagative manner in nature. Here, we show that RBSDV major outer capsid protein (also known as P10) suppresses the protein kinase C (PKC) activity of SBPH through interacting with the receptor for activated protein kinase C 1 (LsRACK1). The N terminal of P10 (amino acids (aa) 1–270) and C terminal of LsRACK1 (aa 268–315) were mapped as crucial for the interaction. Confocal microscopy and subcellular fractionation showed that RBSDV P10 fused to enhanced green fluorescent protein formed vesicular structures associated with endoplasmic reticulum (ER) membranes in Spodoptera frugiperda nine cells. Our results also indicated that RBSDV P10 retargeted the initial subcellular localization of LsRACK1 from cytoplasm and cell membrane to ER and affected the function of LsRACKs to activate PKC. Inhibition of RACK1 by double stranded RNA-induced gene silencing significantly promoted the replication of RBSDV in SBPH. In addition, the PKC pathway participates in the antivirus innate immune response of SBPH. This study highlights that RACK1 negatively regulates the accumulation of RBSDV in SBPH through activating the PKC signalling pathway, and RBSDV P10 changes the subcellular localization of LsRACK1 and affects its function to activate PKC. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

scholarly journals Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis

1998 ◽  
Vol 330 (1) ◽  
pp. 121-129 ◽  
Author(s):  
C. Sarah WOODCOCK ◽  
Evelyne RAUX ◽  
Florence LEVILLAYER ◽  
Claude THERMES ◽  
Alain RAMBACH ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Catalytic Domain ◽  
Conserved Residues ◽  
Pseudomonas Denitrificans ◽  
E Coli ◽  
Cobalamin Biosynthesis ◽  
N Terminus ◽  
Low Levels ◽  
Cobyric Acid

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-L-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

scholarly journals The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

1996 ◽  
Vol 133 (2) ◽  
pp. 269-280 ◽  
Author(s):  
J M Dyer ◽  
J A McNew ◽  
J M Goodman
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Hydrophilic Loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

scholarly journals The PduL Phosphotransacylase Is Used To Recycle Coenzyme A within the Pdu Microcompartment

2015 ◽  
Vol 197 (14) ◽  
pp. 2392-2399 ◽  
Author(s):  
Yu Liu ◽  
Julien Jorda ◽  
Todd O. Yeates ◽  
Thomas A. Bobik
Keyword(s):  
Carbon Fixation ◽  
Coenzyme A ◽  
Fluorescent Protein ◽  
Heterologous Proteins ◽  
Content Type ◽  
Bacterial Microcompartment ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Global Carbon ◽  
Bioinformatic Approach

ABSTRACTInSalmonella enterica, 1,2-propanediol (1,2-PD) utilization (Pdu) is mediated by a bacterial microcompartment (MCP). The Pdu MCP consists of a multiprotein shell that encapsulates enzymes and cofactors for 1,2-PD catabolism, and its role is to sequester a reactive intermediate (propionaldehyde) to minimize cellular toxicity and DNA damage. For the Pdu MCP to function, the enzymes encapsulated within must be provided with a steady supply of substrates and cofactors. In the present study, Western blotting assays were used to demonstrate that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that the N-terminal 20-residue-long peptide of PduL is necessary and sufficient for targeting PduL and enhanced green fluorescent protein (eGFP) to the lumen of the Pdu MCP. We present the results of genetic tests that indicate that PduL plays a role in the recycling of coenzyme A internally within the Pdu MCP. However, the results indicate that some coenzyme A recycling occurs externally to the Pdu MCP. Hence, our results support a model in which a steady supply of coenzyme A is provided to MCP lumen enzymes by internal recycling by PduL as well as by the movement of coenzyme A across the shell by an unknown mechanism. These studies expand our understanding of the Pdu MCP, which has been linked to enteric pathogenesis and which provides a possible basis for the development of intracellular bioreactors for use in biotechnology.IMPORTANCEBacterial MCPs are widespread organelles that play important roles in pathogenesis and global carbon fixation. Here we show that the PduL phosphotransacylase is a component of the Pdu MCP. We also show that PduL plays a key role in cofactor homeostasis by recycling coenzyme A internally within the Pdu MCP. Further, we identify a potential N-terminal targeting sequence using a bioinformatic approach and show that this short sequence extension is necessary and sufficient for directing PduL as well as heterologous proteins to the lumen of the Pdu MCP. These findings expand our general understanding of bacterial MCP assembly and cofactor homeostasis.

scholarly journals Interactions of the TGB1 Protein during Cell-to-Cell Movement of Barley Stripe Mosaic Virus

2001 ◽  
Vol 75 (18) ◽  
pp. 8712-8723 ◽  
Author(s):  
Diane M. Lawrence ◽  
A. O. Jackson
Keyword(s):  
Subcellular Localization ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Triple Gene Block ◽  
Cell Movement ◽  
Barley Stripe Mosaic Virus ◽  
Helicase Domain ◽  
Chenopodium Amaranticolor ◽  
Gene Block ◽  
Green Fluorescent

ABSTRACT We have recently used a green fluorescent protein (GFP) fusion to the γb protein of Barley stripe mosaic virus (BSMV) to monitor cell-to-cell and systemic virus movement. The γb protein is involved in expression of the triple gene block (TGB) proteins encoded by RNAβ but is not essential for cell-to-cell movement. The GFP fusion appears not to compromise replication or movement substantially, and mutagenesis experiments demonstrated that the three most abundant TGB-encoded proteins, βb (TGB1), βc (TGB3), and βd (TGB2), are each required for cell-to-cell movement (D. M. Lawrence and A. O. Jackson, Mol. Plant Pathol. 2:65–75, 2001). We have now extended these analyses by engineering a fusion of GFP to TGB1 to examine the expression and interactions of this protein during infection. BSMV derivatives containing the TGB1 fusion were able to move from cell to cell and establish local lesions in Chenopodium amaranticolor and systemic infections of Nicotiana benthamiana and barley. In these hosts, the GFP-TGB1 fusion protein exhibited a temporal pattern of expression along the advancing edge of the infection front. Microscopic examination of the subcellular localization of the GFP-TGB1 protein indicated an association with the endoplasmic reticulum and with plasmodesmata. The subcellular localization of the TGB1 protein was altered in infections in which site-specific mutations were introduced into the six conserved regions of the helicase domain and in mutants unable to express the TGB2 and/or TGB3 proteins. These results are compatible with a model suggesting that movement requires associations of the TGB1 protein with cytoplasmic membranes that are facilitated by the TGB2 and TGB3 proteins.

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