A Soluble Pyrophosphatase, a Key Enzyme for Polyphosphate Metabolism in Leishmania

Espiau, Benoît; Lemercier, Guillaume; Ambit, Audrey; Bringaud, Frédéric; Merlin, Gilles; Baltz, Théo; Bakalara, Norbert

doi:10.1074/jbc.m506947200

Cited by 40 publications

(51 citation statements)

References 55 publications

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“…This phenomenon seems to be contradictory to high-level expression of H + -PPase in the companion cells. This difference may be due to the difference in molecular activity between PPase and H + -PPase because the molecular activity of PPase is 10-fold higher that of the H + -PPase, although their K m values for PPi are comparable (Nakanishi et al, 2003;Espiau et al, 2006). High activity of E. coli PPase in the transformed companion cells may cause PPi deficiency and suppress hydrolysis of sucrose.…”

Section: Marked Accumulation Of H + -Ppase In Specific Tissuesmentioning

confidence: 95%

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein inArabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

et al. 2014

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We prepared Arabidopsis thaliana lines expressing a functional green fluorescent protein (GFP)-linked vacuolar H + -pyrophosphatase (H + -PPase) under the control of its own promoter to investigate morphological dynamics of vacuoles and tissue-specific expression of H + -PPase. The lines obtained had spherical structures in vacuoles with strong fluorescence, which are referred to as bulbs. Quantitative analyses revealed that the occurrence of the bulbs correlated with the amount of GFP. Next, we prepared a construct of H + -PPase linked with a nondimerizing GFP (mGFP); we detected no bulbs. These results indicate that the membranes adhere face-to-face by antiparallel dimerization of GFP, resulting in the formation of bulbs. In plants expressing H + -PPase-mGFP, intravacuolar spherical structures with double membranes, which differed from bulbs in fluorescence intensity and intermembrane spacing, were still observed in peripheral endosperm, pistil epidermis and hypocotyls. Four-dimensional imaging revealed the dynamics of formation, transformation, and disappearance of intravacuolar spherical structures and transvacuolar strands in living cells. Visualization of H + -PPase-mGFP revealed intensive accumulation of the enzyme, not only in dividing and elongating cells but also in mesophyll, phloem, and nectary cells, which may have high sugar content. Dynamic morphological changes including transformation of vacuolar structures between transvacuolar strands, intravacuolar sheet-like structures, and intravacuolar spherical structures were also revealed.

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Section: Marked Accumulation Of H + -Ppase In Specific Tissuesmentioning

confidence: 95%

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein inArabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

et al. 2014

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“…There are other reports on a role for acidocalcisomes in the virulence of Leishmania. For example, Leishmania mutants of polyphosphate-regulating soluble pyrophosphatase (Espiau et al, 2006), although readily invading macrophages in vitro, were unable to produce lesions in mice. One can speculate that acidocalcisomes play a role in countering environmental changes, such as osmotic stress, that certainly occur during the establishment of an infection.…”

Section: ⌬Ap3␦ Promastigotes Lack Acidocalcisomal Membrane Proteinsmentioning

confidence: 99%

“…The soluble acidocalcisomal protein VSP1 is found in acidocalcisomes of ⌬ap3␦ promastigotes VSP1 is a soluble pyrophosphatase found in T. brucei (Lemercier et al, 2004) and L. amazonensis (Espiau et al, 2006) acidocalcisomes. The ~50-kDa VSP1 was detected in L. major wildtype, AP3␦-null mutant and AP3␦-re-expressing cell extracts with an anti-VSP1 antibody (Fig.…”

Section: ⌬Ap3␦ Promastigotes Lack Acidocalcisomal Membrane Proteinsmentioning

confidence: 99%

“…For immunofluorescence analysis, cells were processed as described previously (Besteiro et al, 2004). Primary antibodies were incubated at the appropriate dilution, 1:1000 for rabbit anti-TbV-H + -PP (Lemercier et al, 2002) and 1:200 for rabbit antiTbVSP1 (Espiau et al, 2006), in PBS with 0.1% BSA for 30 minutes, followed by three washes in PBS and subsequent incubation with fluorochrome-conjugated secondary antibodies. Cells were viewed with a Zeiss UV Axioskop 2 microscope and images were captured by an Orca-ER camera (Hamamatsu) and Openlab software v 5 (Improvision) and deconvolved using the volume deconvolution module.…”

Section: Western Blottingmentioning

confidence: 99%

“…Western blot analysis was performed on cell fractions or total cell extracts (from 10 7 promastigotes) with the anti-TbVP1 rabbit antibody (Lemercier et al, 2002) and rabbit anti-TbVSP1 (Espiau et al, 2006;Lemercier et al, 2004) diluted at 1:1000. Other western blot analyses involved the use of rabbit anti-HASP antibody (Flinn et al, 1994) at a 1:1000 dilution and mouse anti-TbEF1␣ antibody (Upstate) at 1:10,000.…”

Section: Western Blottingmentioning

confidence: 99%

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The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania

Besteiro

Tonn

Tetley

et al. 2008

Journal of Cell Science

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Lysosomal function is crucial for the differentiation and infectivity of the parasitic protozoon Leishmania major. To study lysosomal biogenesis, an L. major mutant deficient in the ␦ subunit of the adaptor protein 3 (AP3 ␦) complex was generated. Structure and proteolytic capacity of the lysosomal compartment were apparently unaffected in the AP3-deficient mutant; however, defects were identified in its acidocalcisomes. These are acidic organelles enriched in calcium and phosphorus, conserved from bacteria to eukaryotes, whose function remains enigmatic. The acidocalcisomes of the L. major mutant lacked membrane-bound proton pumps (notably V-H + -PPase), were less acidic than normal acidocalcisomes and devoid of polyphosphate, but contained a soluble pyrophosphatase. The mutant parasites were viable in vitro, but were unable to establish an infection in mice, which indicates a role for AP3 in determining -possibly through an acidocalcisome-related function -the virulence of the parasite. AP3 transport function has been linked previously to lysosome-related organelles such as platelet dense granules, which appear to share several features with acidocalcisomes. Our findings, implicating that AP3 has a role in transport to acidocalcisomes, thus provide further evidence that biogenesis of acidocalcisomes resembles that of lysosome-related organelles, and that both may have conserved origins. Supplementary material available online at

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Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism

Mirra¹,

Carvalho²,

Curitiba³

et al. 2019

Arch Insect Biochem Physiol

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Polyphosphates (polyPs) have been found in all cell types examined to date and play diverse roles, depending on the cell type. In eukaryotic organisms, polyPs have been mainly investigated in mammalian cells, with few studies on insects.In this study, we investigated mitochondrial polyphosphate metabolism in the red flour beetle, Tribolium castaneum.Substrate specificity for different chain lengths demonstrated the presence of two exopolyphosphatase isoforms in mitochondria. T. castaneum mitochondrial polyP levels decreased after injection with soluble pyrophosphatase (Tc-sPPase) dsRNA, while the membrane exopolyphosphate activity increased. Mitochondrial respiration modulated exopolyphosphatase activity only in wild-type beetles.Tripolyphosphate was able to increase the F-ATPase activity in wild-type and Tc-sPPase RNAi beetles. We suggest that inorganic pyrophosphatase modulates polyphosphate metabolism in mitochondria and affects the link between mitochondrial activity and polyphosphate metabolism in T. castaneum. K E Y W O R D Sinsect, metabolism, mitochondria, polyphosphate

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A Soluble Pyrophosphatase, a Key Enzyme for Polyphosphate Metabolism in Leishmania

Abstract: We report the functional characterization in Leishmania amazonensis of a soluble pyrophosphatase (LaVSP1) that localizes in acidocalcisomes, a vesicular acidic compartment.

Cited by 40 publications

References 55 publications

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein inArabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

Dynamics of Vacuoles and H+-Pyrophosphatase Visualized by Monomeric Green Fluorescent Protein inArabidopsis: Artifactual Bulbs and Native Intravacuolar Spherical Structures

The AP3 adaptor is involved in the transport of membrane proteins to acidocalcisomes of Leishmania

Inorganic pyrophosphatase from the red flour beetle (Tribolium castaneum) modulates mitochondrial polyphosphate metabolism

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