Trypanosoma brucei Mitochondrial Ribosomes

Abstract

Although eukaryotic mitochondrial (mt) ribosomes evolved from a putative prokaryotic ancestor their compositions vary considerably among organisms. We determined the protein composition of tandem affinity-purified Trypanosoma brucei mt ribosomes by mass spectrometry and identified 133 proteins of which 77 were associated with the large subunit and 56 were associated with the small subunit. Comparisons with bacterial and mammalian mt ribosomal proteins identified T. brucei mt homologs of L2-4, L7/12, L9, L11, L13-17, L20 -24, L27-30, L33, L38, L43, L46, L47, L49, L52, S5, S6, S8, S9, S11, S15-18, S29, and S34, although the degree of conservation varied widely. Sequence characteristics of some of the component proteins indicated apparent functions in rRNA modification and processing, protein assembly, and mitochondrial metabolism implying possible additional roles for these proteins. Nevertheless most of the identified proteins have no homology outside Kinetoplastida implying very low conservation and/or a divergent function in kinetoplastid mitochondria. Molecular & Cellular Proteomics 7:1286 -1296, 2008. Mt1 ribosomes appear to be structurally variable to a considerable extent from one organism to another and have undergone some major remodeling during their evolution including loss of RNA and acquisition of proteins (1, 2). Their sedimentation coefficient (S) values vary between 50 S in Leishmania, 70 -74 S in fungi, 55 S in metazoans, and 78 S in higher plants (3-6). Tandem mass spectrometry has been successfully applied to the analysis of the protein composition of ribosomes from several organelles including bovine mitochondria (5, 7-10), fungal mitochondria (11, 12), and chloroplasts (13,14). Mammalian ribosomes have about the same mass as bacterial ribosomes but half as much RNA and over twice as much proteins. Proteomics analysis of bovine mt ribosomes (9, 10) identified 78 different proteins, which include homologs of Escherichia coli ribosomal proteins and 36 additional proteins that presumably were acquired during eukaryotic evolution. Many of these new ribosomal proteins are distinct, having no closely related homologs in bacterial or eukaryotic cytoplasmic ribosomes. The estimated number of proteins within mammalian mt ribosomes has ranged from about 85 to more than 100, for yeast mitochondria it is 78 proteins, and for chloroplast mitochondria it is 49 proteins. The feature of protein richness implies that the reduced rRNA sizes are compensated by some protein components that might have been recruited to mt ribosomes during the course of evolution (15).Trypanosoma and Leishmania are protozoan parasites belonging to the order Kinetoplastida that are the causative agents of several devastating tropical diseases such as African sleeping sickness, Chagas disease, and leishmaniasis. Protein synthesis in the mitochondria of these unicellular flagellates has unusual features. The transcripts from most genes encoded in the trypanosomatid mt DNA are posttranscriptionally modified via extensive and precise ins...

REH2 RNA Helicase in Kinetoplastid Mitochondria

“…S8), and their association was bridged by RNA. We found a few ribosomal proteins in reported TAP purifications of GRBP, KPAP1, MERS1, MRP, TbRGG1, and editing proteins (21, 23), although this had passed unnoticed as the composition of mitochondrial ribosomes was reported after these studies (47). Furthermore, Osato et al (55) found rRNA in TAP isolations of editing complexes.…”

“…8). Interestingly, S2 was not found in isolated mitochondrial ribosomes of T. brucei (47), and we speculate that this protein may help to functionally link MRB-related complexes with ribosomes. Finally, the multicomponent networks we propose may be similar to the L*b complexes recently detected in Leishmania using blue native gel electrophoresis by Osato et al (55), and which the authors proposed represent the active holoenzyme or "editosome.…”

“…In fact, affinity purification of this protein contained REH2, helicase activity, and RECC subunits. 5 Finally, REH2 co-purified with most known subunits of both RNA editing complexes and mitochondrial ribosomes (4,47), largely via RNase-sensitive associations (supplemental Figs. S7 and S8).…”

From simple to supercomplex: mitochondrial genomes of euglenozoan protists

“…RNA editing in Trypanosoma and related flagellates is extremely complex, as the insertions and deletions of hundreds and dozens of uridines, respectively, are performed by a multitude of gRNAs and several protein complexes that interact in a highly dynamic manner 28, 38 . Upon the addition of complex poly-U/A tails 27, 28 ( Figure 4), the fully edited transcripts are translated on protein-rich and rRNA-poor ribosomes 39 , but the role of the additional 45S ribosomal subunit of unique protein composition is still unclear 40 . Likely owing to their extreme hydrophobicity, only a few of the de novo synthesized mt proteins have been observed 41, 42 .…”

The Fe/S Cluster Assembly Protein Isd11 Is Essential for tRNA Thiolation in Trypanosoma brucei

“…Subcellular localization of the expressed tagged proteins within the cell was determined by IFA using polyclonal anti-Myc antibodies (Invitrogen) as described previously (29). Co-localization analysis was performed using monoclonal antibody mAB61 against the MRP1/2 complex (30) coupled with Texas Red X-conjugated secondary antibody (Invitrogen).…”