In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae

Abstract

In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.

Annealing of RNA editing substrates facilitated by guide RNA-binding protein gBP21

“…The insect (procyclic) life cycle stage of T.brucei strain 427 (Cross, 1975) was grown at 27°C in SDM‐79 medium supplemented with 10% (v/v) heat‐inactivated bovine fetal calf serum (Brun and Schönenberger, 1979). Mitochondrial vesicles were isolated by nitrogen cavitation (Hauser et al ., 1996). Detergent lysates of the vesicle preparations were prepared as described by Göringer et al .…”

Snapshots of the RNA editing machine in trypanosomes captured at different assembly stages in vivo

“…Tet-induced insect stage 29-13-MP42/TAP trypanosomes showed a cell-doubling time identical to the parental 29-13 strain and were morphologically indistinguishable from wild-type cells (data not shown). Expression of TbMP42/TAP was verified by Western blotting (Figure 1A) and RNA editing complexes of tet-induced 29-13-MP42/TAP trypanosomes were enriched from non-ionic detergent lysates of mitochondrial vesicles isolated at isotonic conditions (Go ¨ringer et al, 1994;Hauser et al, 1996).…”

Mapping diversity in African trypanosomes using high resolution spatial proteomics

“…1A ) was developed based on previous work 23 , 28 . Cells were lysed by nitrogen cavitation 10 , 34 . Crude membranes and soluble proteins were then separated on an iodixanol cushion with membranes subsequently collected and pelleted by ultracentrifugation.…”

“…Cells were lysed by nitrogen cavitation (cell disruption vessel model 4639, Parr Instrument Company) in line with other work 28 , 34 , 76 . Briefly, the cell suspension was loaded into the pre-chilled vessel which was charged at 350–850 psi and incubated for 15 min before release of the lysate at 1–2 drops/second.…”