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1. Novel IM‐associated protein Tim54 plays a role in the mitochondrial import of internal signal‐containing proteins in Trypanosoma brucei

   https://doi.org/10.1111/boc.202000054  

   Singha, Ujjal K. ; Tripathi, Anuj ; Smith, Jr., Joseph T. ; Quinones, Linda ; Saha, Aparajita ; Singha, Tanusree ; Chaudhuri, Minu ( November 2020 , Biology of the Cell)

   The translocase of the mitochondrial inner membrane (TIM) imports most of the nucleus‐encoded proteins that are destined for the matrix, inner membrane (IM) and the intermembrane space (IMS).Trypanosoma brucei, the infectious agent for African trypanosomiasis, possesses a unique TIM complex consisting of several novel proteins in association with a relatively conserved protein TbTim17. Tandem affinity purification of the TbTim17 protein complex revealed TbTim54 as a potential component of this complex.

   TbTim54, a trypanosome‐specific IMS protein, is peripherally associated with the IM and is present in a protein complex slightly larger than the TbTim17 complex. TbTim54 knockdown (KD) reduced the import of TbTim17 and compromised the integrity of the TbTim17 complex. TbTim54 KD inhibited thein vitromitochondrial import and assembly of the internal signal‐containing mitochondrial carrier proteins MCP3, MCP5 and MCP11 to a greater extent than TbTim17 KD. Furthermore, TbTim54 KD, but not TbTim17 KD, significantly hampered the mitochondrial targeting of ectopically expressed MCP3 and MCP11. These observations along with our previous finding that the mitochondrial import of N‐terminal signal‐containing proteins like cytochrome oxidase subunit 4 and MRP2 was affected to a greater extent by TbTim17 KD than TbTim54 KD indicating a substrate‐specificity of TbTim54 for internal‐signal containing mitochondrial proteins. In other organisms, small Tim chaperones in the IMS are known to participate in the translocation of MCPs. We found that TbTim54 can directly interact with at least two of the six known small TbTim proteins, TbTim11 and TbTim13, as well as with the N‐terminal domain of TbTim17.

   TbTim54 interacts with TbTim17. It also plays a crucial role in the mitochondrial import and complex assembly of internal signal‐containing IM proteins inT. brucei.

   We are the first to characterise TbTim54, a novel TbTim that is involved primarily in the mitochondrial import of MCPs and TbTim17 inT. brucei.

    

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2. The antagonistic MYB paralogs RH1 and RH2 govern anthocyanin leaf markings in Medicago truncatula

   https://doi.org/10.1111/nph.17097  

   Wang, Chongnan ; Ji, Wenkai ; Liu, Yucheng ; Zhou, Peng ; Meng, Yingying ; Zhang, Pengcheng ; Wen, Jiangqi ; Mysore, Kirankumar S. ; Zhai, Jixian ; Young, Nevin D. ; et al ( December 2020 , New Phytologist)

   Patterned leaf coloration in plants generates remarkable diversity in nature, but the underlying mechanisms remain largely unclear.

   Here, usingMedicago truncatulaleaf marking as a model, we show that the classicM. truncatulaleaf anthocyanin spot trait depends on two R2R3 MYB paralogous regulators, RED HEART1 (RH1) and RH2.

   RH1 mainly functions as an anthocyanin biosynthesis activator that specifically determines leaf marking formation depending on its C‐terminal activation motif. RH1 physically interacts with theM. truncatulabHLH protein MtTT8 and the WDR family member MtWD40‐1, and this interaction facilitates RH1 function in leaf anthocyanin marking formation. RH2 has lost transcriptional activation activity, due to a divergent C‐terminal domain, but retains the ability to interact with the same partners, MtTT8 and MtWD40‐1, as RH1, thereby acting as a competitor in the regulatory complex and exerting opposite effects. Moreover, our results demonstrate that RH1 can activate its own expression and that RH2‐mediated competition can repressRH1expression.

   Our findings reveal the molecular mechanism of the antagonistic gene paralogs RH1 and RH2 in determining anthocyanin leaf markings inM. truncatula, providing a multidimensional paralogous–antagonistic regulatory paradigm for fine‐tuning patterned pigmentation.

    

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3. 14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface

   https://doi.org/10.1038/s41467-018-06194-1  

   Karlberg, Tobias ; Hornyak, Peter ; Pinto, Ana Filipa ; Milanova, Stefina ; Ebrahimi, Mahsa ; Lindberg, Mikael ; Püllen, Nikolai ; Nordström, Axel ; Löverli, Elinor ; Caraballo, Rémi ; et al ( September 2018 , Nature Communications)

   Pseudomonasare a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities ofPseudomonasexotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3β:ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment fromVibrioVis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.

    

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4. Ancient MAPK ERK7 is regulated by an unusual inhibitory scaffold required for Toxoplasma apical complex biogenesis

   https://doi.org/10.1073/pnas.1921245117  

   Back, Peter S. ; O’Shaughnessy, William J. ; Moon, Andy S. ; Dewangan, Pravin S. ; Hu, Xiaoyu ; Sha, Jihui ; Wohlschlegel, James A. ; Bradley, Peter J. ; Reese, Michael L. ( June 2020 , Proceedings of the National Academy of Sciences)

   null (Ed.)

   Apicomplexan parasites use a specialized cilium structure called the apical complex to organize their secretory organelles and invasion machinery. The apical complex is integrally associated with both the parasite plasma membrane and an intermediate filament cytoskeleton called the inner-membrane complex (IMC). While the apical complex is essential to the parasitic lifestyle, little is known about the regulation of apical complex biogenesis. Here, we identify AC9 (apical cap protein 9), a largely intrinsically disordered component of the Toxoplasma gondii IMC, as essential for apical complex development, and therefore for host cell invasion and egress. Parasites lacking AC9 fail to successfully assemble the tubulin-rich core of their apical complex, called the conoid. We use proximity biotinylation to identify the AC9 interaction network, which includes the kinase extracellular signal-regulated kinase 7 (ERK7). Like AC9, ERK7 is required for apical complex biogenesis. We demonstrate that AC9 directly binds ERK7 through a conserved C-terminal motif and that this interaction is essential for ERK7 localization and function at the apical cap. The crystal structure of the ERK7–AC9 complex reveals that AC9 is not only a scaffold but also inhibits ERK7 through an unusual set of contacts that displaces nucleotide from the kinase active site. ERK7 is an ancient and autoactivating member of the mitogen-activated kinase (MAPK) family and its regulation is poorly understood in all organisms. We propose that AC9 dually regulates ERK7 by scaffolding and concentrating it at its site of action while maintaining it in an “off” state until the specific binding of a true substrate. 

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5. The Protein-Protein Interaction Network Reveals a Novel Role of the Signal Transduction Protein PII in the Control of c-di-GMP Homeostasis in Azospirillum brasilense

   https://doi.org/10.1128/mSystems.00817-20  

   Gerhardt, Edileusa C. ; Parize, Erick ; Gravina, Fernanda ; Pontes, Flávia L. ; Santos, Adrian R. ; Araújo, Gillize A. ; Goedert, Ana C. ; Urbanski, Alysson H. ; Steffens, Maria B. ; Chubatsu, Leda S. ; et al ( December 2020 , mSystems)

   Porto, Carla (Ed.)

   ABSTRACT The PII family comprises a group of widely distributed signal transduction proteins ubiquitous in prokaryotes and in the chloroplasts of plants. PII proteins sense the levels of key metabolites ATP, ADP, and 2-oxoglutarate, which affect the PII protein structure and thereby the ability of PII to interact with a range of target proteins. Here, we performed multiple ligand fishing assays with the PII protein orthologue GlnZ from the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense to identify 37 proteins that are likely to be part of the PII protein-protein interaction network. Among the PII targets identified were enzymes related to nitrogen and fatty acid metabolism, signaling, coenzyme synthesis, RNA catabolism, and transcription. Direct binary PII-target complex was confirmed for 15 protein complexes using pulldown assays with recombinant proteins. Untargeted metabolome analysis showed that PII is required for proper homeostasis of important metabolites. Two enzymes involved in c-di-GMP metabolism were among the identified PII targets. A PII-deficient strain showed reduced c-di-GMP levels and altered aerotaxis and flocculation behavior. These data support that PII acts as a major metabolic hub controlling important enzymes and the homeostasis of key metabolites such as c-di-GMP in response to the prevailing nutritional status. IMPORTANCE The PII proteins sense and integrate important metabolic signals which reflect the cellular nutrition and energy status. Such extraordinary ability was capitalized by nature in such a way that the various PII proteins regulate different facets of metabolism by controlling the activity of a range of target proteins by protein-protein interactions. Here, we determined the PII protein interaction network in the plant growth-promoting nitrogen-fixing bacterium Azospirillum brasilense . The interactome data along with metabolome analysis suggest that PII functions as a master metabolic regulator hub. We provide evidence that PII proteins act to regulate c-di-GMP levels in vivo and cell motility and adherence behaviors. 

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