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Trypanosomal Pam18/Pam16 and maxicircle replication – TbPam18 SILAC RNAiTrypanosomal Pam18/Pam16 and maxicircle replication – TbPam18 SILAC RNAi

ABSTRACT: Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the two proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. The level of MaRF11 is controlled by the mitochondrial proteasome. Thus, we propose a model for a membrane-bound regulatory circuit that controls maxicircle replication in response to an unknown nuclear signal. This model posits that MaRF11 directly mediates maxicircle replication, that its activity is regulated by the mitochondrial proteasome that its level is controlled by proteasomal digested that it is protected from degradation by binding to the TbPam18/TbPam16 dimer.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Julian Bender

LAB HEAD: Bettina Warscheid

PROVIDER: PXD046845 | Pride | 2024-07-20

REPOSITORIES: Pride

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Trypanosomal Pam18/Pam16 and maxicircle replication – TbPam16 SILAC RNAi

Project description:Protein import and genome replication are essential processes for mitochondrial biogenesis and propagation. The J-domain proteins Pam16 and Pam18 regulate the presequence translocase of the mitochondrial inner membrane. In the protozoan Trypanosoma brucei, their counterparts are TbPam16 and TbPam18, which are essential for the procyclic form of the parasite, though not involved in mitochondrial protein import. Here, we show that during evolution, the two proteins have been repurposed to regulate the replication of maxicircles within the intricate kDNA network, the most complex mitochondrial genome known. TbPam18 and TbPam16 have inactive J-domains suggesting a function independent of heat shock proteins. However, their single transmembrane domain is essential for function. Pulldown of TbPam16 identifies a putative client protein, termed MaRF11, the depletion of which causes the selective loss of maxicircles, akin to the effects observed for TbPam18 and TbPam16. The level of MaRF11 is controlled by the mitochondrial proteasome. Thus, we propose a model for a membrane-bound regulatory circuit that controls maxicircle replication in response to an unknown nuclear signal. This model posits that MaRF11 directly mediates maxicircle replication, that its activity is regulated by the mitochondrial proteasome that its level is controlled by proteasomal digested that it is protected from degradation by binding to the TbPam18/TbPam16 dimer.

2024-07-20 | PXD046840 | Pride

Trypanosomal Pam18/Pam16 and maxicircle replication – MaRF11 SILAC RNAi

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Release of a nuclear ubiquitin-like protein activates proteasomal degradation of mislocalized hydrophobic mitochondrial proteins in trypanosomes

Project description:Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential since cytosolic TbUbL1 can bind mislocalized mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all mitochondria.

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Release of a nuclear ubiquitin-like protein activates proteasomal degradation of mislocalized hydrophobic mitochondrial proteins in trypanosomes

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Trypanosomal Pam18/Pam16 and maxicircle replication – TbPam16 CoIPs

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Charting Organellar Importomes by Quantitative Mass Spectrometry – PEX14c

Project description:Protein import into organelles is essential for all eukaryotes and facilitated by multi-protein translocation machineries. Analyzing whether a protein is transported into an organelle is largely restricted to single constituents. This renders knowledge about imported proteins incomplete, limiting our understanding of organellar biogenesis and function. Here, we introduce a method that enables to chart an organelle’s importome. Our method relies on inducible RNAi-mediated knockdown of an essential subunit of a translocase to impair import, metabolic labeling and quantitative mass spectrometry. To highlight its potential, we established the mitochondrial importome of Trypanosoma brucei, comprising 1,120 proteins including 331 new candidates. An exciting feature of this method is that it overcomes limitations in identifying proteins with dual or multiple locations. We demonstrate the specificity and versatility of this novel ImportOmics method by targeting import machineries in mitochondria and glycosomes demonstrating its high potential for globally studying protein import and inventories of organelles.

2017-06-14 | PXD004972 | Pride

Replication

Project description:Replication

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The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

Project description:Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.

2016-12-20 | PXD005484 | Pride

Dynamic Patterns of Mammalian Mitochondrial DNA Replication Uncovered Using SSiNGLe-5′ES

Project description:The proper replication of mitochondrial DNA is key to the maintenance of this crucial organelle. Multiple studies aimed at understanding the mechanisms of replication of the mitochondrial genome have been conducted in the past several decades; however, while highly informative, they were conducted using relatively low-sensitivity techniques. Here, we established a high-throughput approach based on next-generation sequencing to identify replication start sites with nucleotide-level resolution and applied it to the genome of mitochondria from different human and mouse cell types. We found complex and highly reproducible patterns of mitochondrial initiation sites, both previously annotated and newly discovered in this work, that showed differences among different cell types and species. These results suggest that the patterns of the replication initiation sites are dynamic and might reflect, in some yet unknown ways, the complexities of mitochondrial and cellular physiology. Overall, this work suggests that much remains unknown about the details of mitochondrial DNA replication in different biological states, and the method established here opens up a new avenue in the study of the replication of mitochondrial and potentially other genomes.

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DSB formation in replication compromised cells

Project description:DSBs were mapped genome-wide by ssDNA enrichment in cdc6-mn replication comprimsied strains. We mapped DSB sites by detecting DSB-associated ssDNA enrichment on microarrays. To test the role of DNA replication in DSB formation, we mapped ssDNA in a cdc6-mn replication depleted strain. ssDNA was isolated from cells after 5 hours in sporulation medium. As a reference, ssDNA isolated from cells at 0 hrs in sporulation medium prior to DSB formation was differentially labeled and co-hybridized to the same array. For each experiment, we have submitted biological replicates that were hybridized to separate arrays. For each experiment a dye swap was performed and shown to have no effect on the data observed, although not all experiments in this series include the dye swap sample (we have included only two representative experiments for each strain).

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