A Scalable Purification Method for Mitochondria from Trypanosoma brucei

Niemann, Moritz; Schneider, André

doi:10.1007/978-1-0716-0294-2_36

Cited by 7 publications

(7 citation statements)

References 26 publications

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“…For large scale preparations of mitochondrial vesicles (see below), cells were grown in 2L Erlenmeyer flasks to a density of up to 6x10 7 cells/mL in a total volume of 1-1.5 L at 27 C and shaking at 120 rpm. The vesicle preparation for small to large scale is described in detail in Niemann and Schneider (2020).…”

Section: Star+methodsmentioning

confidence: 99%

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

et al. 2020

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Section: Star+methodsmentioning

confidence: 99%

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

et al. 2020

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“… 7 and is described in detail in ref. 63 . Briefly, up to 1 × 10 12 cells (density maximally at 6 × 10 7 cells/mL) were harvested by centrifugation, washed with 1× SBG (20 mM sodium phosphate, pH 7.9; 20 mM glucose; 0.15 M NaCl) and lysed by nitrogen cavitation (70 bar, 45 to 60 min) at isotonic conditions using 1× SoTE (600 mM sorbitol, 20 mM Tris⋅HCl pH 7.5, 1 mM ethylenediaminetetraacetic acid).…”

Section: Methodsmentioning

confidence: 99%

“…The lysate was subjected to differential centrifugation and subsequent DNase digestion as detailed in ref. 63 . The mitochondrial vesicles were further purified using density gradient centrifugation in a 18/21/25/28% (wt/vol) Nycodenz step-gradient containing 1× SoTE.…”

Section: Methodsmentioning

confidence: 99%

Mitoribosomal small subunit maturation involves formation of initiation-like complexes

Lenarčič

Niemann

Ramrath

et al. 2022

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

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Mitochondrial ribosomes (mitoribosomes) play a central role in synthesizing mitochondrial inner membrane proteins responsible for oxidative phosphorylation. Although mitoribosomes from different organisms exhibit considerable structural variations, recent insights into mitoribosome assembly suggest that mitoribosome maturation follows common principles and involves a number of conserved assembly factors. To investigate the steps involved in the assembly of the mitoribosomal small subunit (mt-SSU) we determined the cryoelectron microscopy structures of middle and late assembly intermediates of the Trypanosoma brucei mitochondrial small subunit (mt-SSU) at 3.6- and 3.7-Å resolution, respectively. We identified five additional assembly factors that together with the mitochondrial initiation factor 2 (mt-IF-2) specifically interact with functionally important regions of the rRNA, including the decoding center, thereby preventing premature mRNA or large subunit binding. Structural comparison of assembly intermediates with mature mt-SSU combined with RNAi experiments suggests a noncanonical role of mt-IF-2 and a stepwise assembly process, where modular exchange of ribosomal proteins and assembly factors together with mt-IF-2 ensure proper 9S rRNA folding and protein maturation during the final steps of assembly.

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“…The detailed procedure for the purification of mitochondrial vesicles from a variety of different cell amounts is detailed eslewhere 47 . Briefly, trypanosomes were lysed at isotonic conditions using nitrogen cavitation, and following differential centrifugation, mitochondrial vesicles were purified from Nycodenz step-gradients subjected to density gradient centrifugation.…”

Section: Preparation Of Mitochondrial Vesicles Mitochondrial Outer Membrane (Om) and Om/er Fractionsmentioning

confidence: 99%

“…To establish the sub-organellar localization of PSS2 in T. brucei, we generated a cell line expressing TbPSS2 (Tb927.7.3760) containing a C-terminal cMyc-tag from an ectopic locus under the control of tetracycline and executed a well-established cell fractionation protocol to generate highly purified mitochondrial vesicles (Fig. 8A) 47 . The resultant fractions were analyzed by immunoblotting and probed for cMyc and the MOM marker ATOM40.…”

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The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei

Iyer

Niemann

Serricchio

et al. 2021

Preprint

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The endoplasmic reticulum membrane complex (EMC) is a versatile complex that plays a key role in membrane protein biogenesis in the ER. Deletion of the complex has wide-ranging consequences including ER stress, disturbance in lipid transport and organelle tethering, among others. Here we report the function and organization of the evolutionarily conserved EMC (TbEMC) in the highly diverged eukaryote, Trypanosoma brucei. We show that the TbEMC is composed of 9 subunits that are present in a high molecular mass complex localizing to the mitochondrial-endoplasmic reticulum interface. Knocking out or knocking down of single TbEMC subunits led to growth defects of T. brucei procyclic forms in culture. Interestingly, we found that depletion of individual TbEMC subunits lead to disruption of de novo synthesis of phosphatidylcholine (PC) or phosphatidylethanolamine (PE), the two most abundant phospholipid classes in T. brucei . Downregulation of TbEMC1 or TbEMC3 inhibited formation of PC while depletion of TbEMC8 inhibited PE synthesis, re-emphasizing the role of the EMC in phospholipid synthesis. In addition, we found that in TbEMC7 knock-out parasites, TbEMC3 is released from the complex, implying that TbEMC7 is essential for the formation or the maintenance of the TbEMC. The organization of the TbEMC and its role in phospholipid metabolism in T. brucei may represent ancestral functions.

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A Scalable Purification Method for Mitochondria from Trypanosoma brucei

Cited by 7 publications

References 26 publications

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

Structural Insights into the Mechanism of Mitoribosomal Large Subunit Biogenesis

Mitoribosomal small subunit maturation involves formation of initiation-like complexes

The endoplasmic reticulum membrane protein complex localizes to the mitochondrial - endoplasmic reticulum interface and its subunits modulate phospholipid biosynthesis in Trypanosoma brucei

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