Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

Chen, Allan L.; Kim, Elliot W.; Toh, Justin Y.; Vashisht, Ajay A.; Rashoff, Andrew Q.; Van, Christina; Huang, Amy S.; Moon, Andy S; Bell, Hannah; Bentolila, Laurent A.; Wohlschlegel, James A.; Bradley, Peter J.

doi:10.1128/mbio.02357-14

Cited by 163 publications

(209 citation statements)

References 57 publications

(106 reference statements)

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“…To confirm that IMC proteins are modified by ubiquitin, we performed immunoprecipitation (IP) of epitope-tagged IMC proteins (Beck et al, 2010; Chen et al, 2014) from intracellular tachyzoites followed by Western blot using anti-ubiquitin antibodies (Figure 4A). To prevent degradation of polyubiquitinated proteins, intracellular parasites were treated with the proteasome inhibitor MG-132 before lysate preparation (Shaw et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

Monerri

Yakubu

Chen

et al. 2015

Cell Host & Microbe

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Protein ubiquitination plays key roles in protein turnover, cellular signalling, and intracellular transport. The genome of Toxoplasma gondii encodes ubiquitination machinery but the roles of this posttranslational modification (PTM) are unknown. To examine the prevalence and function of ubiquitination in T. gondii, we mapped the ubiquitin proteome of tachyzoites. Over 500 ubiquitin-modified proteins, with almost 1000 sites, were identified on proteins with diverse localisations and functions. Enrichment analysis demonstrated that 35% of ubiquitinated proteins are cell cycle-regulated. Unexpectedly, most classic cell cycle regulators conserved in T. gondii were not detected in the ubiquitinome. Furthermore, many ubiquitinated proteins localise to the cytoskeleton and inner membrane complex, a structure beneath the plasma membrane facilitating division and host invasion. Comparing the ubiquitinome with other PTM proteomes reveals waves of PTM enrichment during the cell cycle. Thus, T. gondii PTMs are implicated as critical regulators of cell division and cell cycle transitions.

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Section: Resultsmentioning

confidence: 99%

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

Monerri

Yakubu

Chen

et al. 2015

Cell Host & Microbe

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“…To avoid division by 0 we used 0.2 as an arbitrary value in those cases where no trace of the protein was detected in the HA- fraction. This list includes proteins already described to be palmitoylated (Table S1 highlighted in blue) such as HSP20 [12], GAP45 and AMA1 [15] and members of the ISP family [9], those predicted to be palmitoylated but without experimental evidence (Table S1 highlighted in grey) such as members of the ISC (IMC structure component), IMC (inner membrane complex) and AC (apical cap) families [32], and those that share homology to known S-acylated proteins from Plasmodium falciparum , a related organism, such as 14-3-3, Hsp70 and proteins of the CDPK family [33] (Table S1 highlighted in green). We also identified TGME49_213550 (TgDHHC 4), TGME49_249380 (TgDHHC13) and TGME49_278850 (TgDHHC2) which are DHHC S-acyl transferases [18].…”

Section: Resultsmentioning

confidence: 99%

“…Regarding function and biological processes in which palmitoylated proteins are involved, most of them cluster into groups including metabolic and energy-related processes and protein translation (Figure 2B). However, it is evident that the modification affects the majority of cellular functions since we were able to identify proteins involved in a plethora of roles: from signaling (CDPK3), to gliding motility (myosin A, GAP45 and various filament-like alveolins, members of the recently described IMC, AC and ISC families [32]), translation (translation initiation factor eIF-5A and translation elongation factor 2), among others. Some of these proteins have been previously reported to be palmitoylated: in the case of TgCDPK3, studies have shown that palmitoylation not only occurs but it also affects the localization of the protein and thus its function in the parasite’s ability to egress the host-cell [35].…”

Section: Resultsmentioning

confidence: 99%

Identification of new palmitoylated proteins in Toxoplasma gondii

Caballero

Alonso

Deng

et al. 2016

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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Protein palmitoylation has been shown to be an important post-translational modification in eukaryotic cells. This modification alters the localization and/or the function of the targeted protein. In the recent years protein palmitoylation has risen in importance in apicomplexan parasites as well. In Toxoplasma gondii, some proteins have been reported to be modified by palmitate. With the development of new techniques that allow the isolation of palmitoylated proteins, this significant post-translational modification has begun to be studied in more detail in T. gondii. Here we describe the palmitoylome of the tachyzoite stage of T. gondii using a combination of the acyl-biotin exchange chemistry method and mass spectrometry analysis. We identified 401 proteins found in multiple cellular compartments, with a wide range of functions that vary from metabolic processes, gliding and host-cell invasion to even regulation of transcription and translation. Besides, we found that more rhoptry proteins than the ones already described for Toxoplasma are palmitoylated, suggesting an important role for this modification in the invasion mechanism of the host-cell. This study documents that protein palmitoylation is a common modification in T. gondii that could have an impact on different cellular processes. Table S1: List of all non-redundant identified candidate proteins from the three independent ABE assays. The first worksheet tab contains a multiconsensus analysis showing proteins identified in all three assays together with their respective mass spectrometry information and in silico predictions. Highlighted in blue are those already described to palmitoylated, in grey are those predicted to be palmitoylated but without experimental evidence and in green those that have homology to known S-acylated proteins from P. falciparum. Tabs 2, 3 and 4 contain particular information and NRP calculations for each assay, while the last tab is a summary of the calculated NRP HA+/HA-ratios calculated for each protein.

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“…The method also has been used to screen for proteins involved in the Hippo signaling pathway (Couzens et al, 2013). Additionally, BioID has been applied to a secreted protein of Chlamydia psittaci that targets the nuclear envelope (Mojica et al, 2015), used to identify novel components of the inner membrane complex of Toxoplasma gondii (Chen et al, 2015), analyzing HIV-1 Gag protein interactions (Ritchie et al, 2015), and utilized in detecting c-MYC interacting partners in cultured cells and mice xenograft tumors (Dingar et al, 2014). …”

Section: Introductionmentioning

confidence: 99%

BioID Identification of Lamin-Associated Proteins

Mehus

Anderson

Roux

2016

Methods in Enzymology

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The nuclear lamina is primarily composed of type-V intermediate filaments, the A- and B-type lamins, which give mechanical support to the nuclear envelope, contribute to heterochromatin organization and regulate a myriad of nuclear processes. Over a dozen human diseases, collectively named laminopathies, are associated with mutations in the genes that encode the nuclear lamins. Although the etiology of the laminopathies is well understood, the molecular mechanisms by which they lead to disease remain unclear. Also poorly understood are the mechanisms by which the lamins contribute to a variety of nuclear and cellular functions. The identification of proteins associated with the lamins is likely to provide insight into these fundamental mechanisms. In recent years a unique method for identifying protein-protein and proximity-based interactions has emerged, called BioID (proximity-dependent biotin identification). BioID utilizes a mutant biotin ligase from bacteria that is fused to a protein of interest (bait). When expressed in living cells and stimulated with excess biotin, this BioID fusion protein promiscuously biotinylates directly interacting and vicinal endogenous proteins. Following biotin-affinity capture, the biotinylated proteins can be identified using mass spectrometry (MS). BioID thus enables screening for physiologically relevant protein associations that occur over time in living cells. The application of BioID is amenable to insoluble proteins such as lamins that are often refractory to study by other methods and is capable of identifying weak and/or transient interactions. In this review, we discuss the use of BioID as a powerful tool to help elucidate novel interacting partners of lamins.

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Novel Components of the Toxoplasma Inner Membrane Complex Revealed by BioID

Cited by 163 publications

References 57 publications

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

The Ubiquitin Proteome of Toxoplasma gondii Reveals Roles for Protein Ubiquitination in Cell-Cycle Transitions

Identification of new palmitoylated proteins in Toxoplasma gondii

BioID Identification of Lamin-Associated Proteins

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