Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Huang, Guozhong; Ulrich, Paul N.; Storey, Melissa; Johnson, David; Tischer, Julie; Tovar, Javier A.; Moreno, Silvia N.J.; Orlando, Ron; Docampo, Roberto

doi:10.1371/journal.ppat.1004555

Cited by 82 publications

(126 citation statements)

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“…This perhaps represents a partial compensation, in which energy is stored in phosphoanhydride bonds of inositol pyrophosphates rather than polyP. It is clear that the additional energy stored in the phosphoanhydride bonds of InsP 7 and InsP 8 in ppk1 will not be equal to that accumulated in WT polyP. However, these results do confirm a metabolic interplay between these two families of phosphate-rich molecules and their ability to regulate or be regulated by cellular energy metabolism.…”

Section: Altered Atp and Accumulation Of Inositol Pyrophosphates In Pmentioning

confidence: 85%

“…The analysis of inositol phosphates extracted from WT and ppk1 cells revealed that the ratio of InsP 7 and InsP 8 to their precursor InsP 6 is significantly altered (Fig. 5A).…”

Section: Altered Atp and Accumulation Of Inositol Pyrophosphates In Pmentioning

confidence: 99%

“…However, polyP is also present in archaea (3), eukaryotes (4), and human cells (5). Whilst polyP is present in virtually all cellular compartments (6), it accumulates in acidocalcisomes, organelles common to bacteria and mammalian cells (7).…”

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confidence: 99%

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Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Livermore

Chubb

Saiardi

2016

Proc. Natl. Acad. Sci. U.S.A.

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Inorganic polyphosphate (polyP) is composed of linear chains of phosphate groups linked by high-energy phosphoanhydride bonds. However, this simple, ubiquitous molecule remains poorly understood. The use of nonstandardized analytical methods has contributed to this lack of clarity. By using improved polyacrylamide gel electrophoresis we were able to visualize polyP extracted from Dictyostelium discoideum. We established that polyP is undetectable in cells lacking the polyphosphate kinase (DdPpk1). Generation of this ppk1 null strain revealed that polyP is important for the general fitness of the amoebae with the mutant strain displaying a substantial growth defect. We discovered an unprecedented accumulation of polyP during the developmental program, with polyP increasing more than 100-fold. The failure of ppk1 spores to accumulate polyP results in a germination defect. These phenotypes are underpinned by the ability of polyP to regulate basic energetic metabolism, demonstrated by a 2.5-fold decrease in the level of ATP in vegetative ppk1. Finally, the lack of polyP during the development of ppk1 mutant cells is partially offset by an increase of both ATP and inositol pyrophosphates, evidence for a model in which there is a functional interplay between inositol pyrophosphates, ATP, and polyP.inorganic polyphosphate | phosphate | metabolism | inositol pyrophosphate | mitochondria T he simplest biological polymer is inorganic polyphosphate (polyP), which consists of a linear chain of phosphates linked by high-energy phosphoanhydride bonds (1). Regardless of its origin, polyP is ubiquitously present in today's living organisms. The synthesis, enzymology, and role of polyP are best characterized in the bacteria Escherichia coli (2). However, polyP is also present in archaea (3), eukaryotes (4), and human cells (5). Whilst polyP is present in virtually all cellular compartments (6), it accumulates in acidocalcisomes, organelles common to bacteria and mammalian cells (7).PolyP has been implicated in a range of cellular processes over recent years, but its most basic proposed function is to act as a buffer for the level of free phosphate (8). Phosphate buffering is essential for general metabolism, and the synthesis or degradation of polyP is able to safeguard the level of free phosphate in cells. In addition, the polyanionic nature of polyP makes it a strong chelator of bivalent cations; thus polyP plays a key role in cation homeostasis. The chelation of calcium by polyP might even play a role as a calcium sink, regulating calcium signaling (8). Besides these common roles, many specific functions have been attributed to polyP in eukaryotes (see reviews in refs. 9-11). Recently its ability to function as a protein-folding chaperone (12) and to drive a newly discovered posttranslational protein modification, protein polyphosphorylation (13), have provided further insight into the mechanism of action of this simple polymer.Little is known about the synthesis or metabolism of polyP in eukaryotes. The yeast Saccharomyces c...

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Section: Altered Atp and Accumulation Of Inositol Pyrophosphates In Pmentioning

confidence: 85%

“…The analysis of inositol phosphates extracted from WT and ppk1 cells revealed that the ratio of InsP 7 and InsP 8 to their precursor InsP 6 is significantly altered (Fig. 5A).…”

Section: Altered Atp and Accumulation Of Inositol Pyrophosphates In Pmentioning

confidence: 99%

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Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Livermore

Chubb

Saiardi

2016

Proc. Natl. Acad. Sci. U.S.A.

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“…TbIP 3 R in T. brucei was demonstrated by endogenous gene tagging (13), by studies using specific antibodies (14), and by proteomic (14) and functional (13) studies. Interestingly, when TbIP 3 R (13) or the IP 3 Rs of Capsaspora owczarzaki (another protist in which biochemical characterization of the channel was done (34)) are expressed in DT40 -3KO cells (chicken lymphocytes in which the three vertebrate IP 3 R have been knocked out), the proteins localize to the ER.…”

Section: Crispr/cas9-mediated Endogenous Tagging In T Cruzimentioning

confidence: 99%

“…However, the immunofluorescence evidence reported was disputed (12), because there was no clear reticular pattern or co-localization with a T. brucei ER marker, TbBiP, in the figures published (11). In addition, the T. brucei IP 3 R localized to the acidocalcisomes as demonstrated using antibodies against the endogenous tagged protein (13) and specific antibodies against the protein (14), as well as proteomic and functional analyses (13,14). In this work, we report the acidocalcisome localization of TcIP 3 R.…”

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confidence: 99%

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor

Lander

Chiurillo

Storey

et al. 2016

Journal of Biological Chemistry

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Edited by Dennis VoelkerMethods for genetic manipulation of Trypanosoma cruzi, the etiologic agent of Chagas disease, have been highly inefficient, and no endogenous tagging of genes has been reported to date. We report here the use of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system for endogenously tagging genes in this parasite. The utility of the method was established by tagging genes encoding proteins of known localization such as TcFCaBP (flagellar calcium binding protein) and TcVP1 (vacuolar proton pyrophosphatase), and two proteins of undefined or disputed localization, the TcMCU (mitochondrial calcium uniporter) and TcIP 3 R (inositol 1,4,5-trisphosphate receptor). We confirmed the flagellar and acidocalcisome localization of TcFCaBP and TcVP1 by co-localization with antibodies to the flagellum and acidocalcisomes, respectively. As expected, TcMCU was colocalized with the voltage-dependent anion channel to the mitochondria. However, in contrast to previous reports and our own results using overexpressed TcIP 3 R, endogenously tagged TcIP 3 R showed co-localization with antibodies against VP1 to acidocalcisomes. These results are also in agreement with our previous reports on the localization of this channel to acidocalcisomes of Trypanosoma brucei and suggest that caution should be exercised when overexpression of tagged genes is done to localize proteins in T. cruzi.The application of the CRISPR/Cas9 technology to the study of protist parasites has dramatically increased the tools available for their genetic manipulation (1). Trypanosoma cruzi, the etiologic agent of Chagas disease, which is a significant cause of morbidity and mortality from the South of the United States to the South of Argentina and Chile, has been particularly refractory to genetic manipulation. However, the recent use of the CRISPR/Cas9 technology to knock down or knock out genes (2, 3) has revolutionized their study.The localization of proteins is important to determine their cellular function, and previous studies in T. cruzi have used either antibodies or gene tagging methods with vectors that overexpressed the proteins (4). Although specific antibodies are useful to detect the endogenous proteins, it is not always possible to obtain them because either the proteins have low antigenicity or the antibodies cross-react with other proteins. Plasmids that enable the tagging of genes at their endogenous loci are not available for T. cruzi, and a major drawback of the overexpression of tagged proteins is that the proteins of interest sometimes are retained in the endoplasmic reticulum (ER) 4 or localize to other compartments.Here we have adapted the CRISPR/Cas9 system to tag genes of T. cruzi at their endogenous loci and tested this system with two genes encoding proteins of well recognized localization (flagellar calcium binding protein or TcFCaBP and the acidocalcisome vacuolar proton pyrophosphatase or TcVP1) and two encoding proteins of undefined or disputed localizati...

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Flagellar membrane proteins in kinetoplastid parasites

2015

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All kinetoplastid parasites, including protozoa such as Leishmania species, Trypanosoma brucei, and Trypanosoma cruzi that cause devastating diseases in humans and animals, are flagellated throughout their life cycles. While flagella were originally thought of primarily as motility organelles, flagellar functions in other critical processes, especially in sensing and signal transduction, have become more fully appreciated in the recent past. The flagellar membrane is a highly specialized subdomain of the surface membrane, and flagellar membrane proteins are likely to be critical components for all the biologically important roles of flagella. In this review, we summarize recent discoveries relevant to flagellar membrane proteins in these parasites including the identification of such proteins, investigation of their biological functions, and mechanisms of selective trafficking to the flagellar membrane. Prospects for future investigations and current unsolved problems are highlighted.

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Proteomic Analysis of the Acidocalcisome, an Organelle Conserved from Bacteria to Human Cells

Cited by 82 publications

References 104 publications

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

Developmental accumulation of inorganic polyphosphate affects germination and energetic metabolism in Dictyostelium discoideum

CRISPR/Cas9-mediated endogenous C-terminal Tagging of Trypanosoma cruzi Genes Reveals the Acidocalcisome Localization of the Inositol 1,4,5-Trisphosphate Receptor

Flagellar membrane proteins in kinetoplastid parasites

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