tRNAs and proteins are imported into mitochondria of Trypanosoma brucei by two distinct mechanisms.

Import of tRNA into the mitochondrial matrix of Trypanosoma brucei was reconstituted in vitro. Efficient import required the hydrolysis of externally added ATP and was shown to be a carrier-mediated process depending on proteinaceous receptors on the surface of mitochondria. A partly synthetic tRNA(Tyr) as well as a physiological tRNA(Lys) were imported along the same pathway. Contrary to import of all matrix-localized proteins, tRNA import does not require a membrane potential. Furthermore, addition of an excess of import-competent tRNA had no effect on import of a mitochondrial matrix protein. In summary, these results show that tRNAs and proteins in T. brucei are imported by fundamentally different mechanisms.

Chloramphenicol-sensitive mitochondrial translation in Trypanosoma brucei.

We developed an in organello system to label newly synthesized mitochondrially encoded proteins of Trypanosoma brucei. Highly purified mitochondria, prepared under isotonic conditions, were incubated with radioactive methionine and cysteine in a suitable translation buffer. Analysis of mitochondrial extracts on TRIS-Tricine gels revealed a subset of labeled, NP-40-insoluble proteins. The labeling of these proteins was resistant to the cytosol-specific translation inhibitor cycloheximide. The proteins, however, were not labeled in the presence of chloramphenicol or erythromycin, inhibitors of prokaryotic type translation, or puromycin, a general translation inhibitor. These results indicate that isotonically isolated mitochondria of T. brucei are capable of protein synthesis.

In vivo import of unspliced tRNATyr containing synthetic introns of variable length into mitochondria of Leishmania tarentolae.

The mitochondrial genomes of trypanosomatids lack tRNA genes. Instead, mitochondrial tRNAs are encoded and synthesized in the nucleus and are then imported into mitochondria. This also applies for tRNATyr, which in trypanosomatids contains an 11 nt intron. Previous work has defined an exon mutation which leads to accumulation of unspliced precursor tRNATyr. In this study we have used the splicing-deficient tRNATyr as a vehicle to introduce foreign sequences into the mitochondrion of Leishmania tarentolae. The naturally occurring intron was replaced by synthetic sequences of increasing length and the resulting tRNATyr precursors were expressed in transgenic cell lines. Whereas stable expression of precursor tRNAsTyr was obtained for introns up to a length of 76 nt, only precursors having introns up to 38 nt were imported into mitochondria. These results demonstrate that splicing-deficient tRNATyr can be used to introduce short synthetic sequences into mitochondria in vivo. In addition, our results show that one factor which limits the efficiency of import is the length of the molecule.

Leishmania tarentolae contains distinct cytosolic and mitochondrial glutaminyl-tRNA synthetase activities.

The intracellular distribution of glutaminyl-tRNA synthetases and their role in mitochondrial tRNA import were evaluated in the ancient eukaryote Leishmania tarentolae. The following results were obtained: (i) Glutaminyl-tRNA synthetase was detected in leishmanial mitochondria. This was unexpected because it has been postulated that, in organelles, Gln-tRNAGln is not formed by direct acylation of tRNAGln but by enzymatic transamidation of misacylated Glu-tRNAGln. (ii) Whereas the cytosolic extract is able to charge cytosolic and mitochondrial tRNAsGln, the mitochondrial matrix extract does not aminoacylate the cytosol-specific tRNAGln. This indicates that mitochondrial and cytosolic glutaminyl-tRNA synthetases are distinct. (iii) Seven of the 11 nucleotides that differ between the cytosolic and the mitochondrial tRNAGln are sufficient to convert the cytosol-specific tRNAGln into an optimal substrate for the mitochondrial enzyme. These nucleotides are arranged in three groups consisting of the nucleotides flanking the anticodon stem, the 5' nucleotide of the anticodon, and four nucleotides within the acceptor stem. And (iv), it was shown that the identity elements for recognition by the mitochondrial glutaminyl-tRNA synthetase do not overlap with a previously identified sequence segment required for mitochondrial import of the tRNAGln.

Transmembrane orientation of signal-anchor proteins is affected by the folding state but not the size of the N-terminal domain.

Upon insertion of a signal-anchor protein into the endoplasmic reticulum membrane, either the C-terminal or the N-terminal domain is translocated across the membrane. Charged residues flanking the transmembrane domain are important determinants for this decision, but are not necessarily sufficient to generate a unique topology. Using a model protein that is inserted into the membrane to an equal extent in either orientation, we have tested the influence of the size and the folding state of the N-terminal domain on the insertion process. A small zinc finger domain or the full coding sequence of dihydrofolate reductase were fused to the N-terminus. These stably folding domains hindered or even prevented their translocation. Disruption of their structure by destabilizing mutations largely restored transport across the membrane. Translocation efficiency, however, did not depend on the size of the N-terminal domain within a range of 40-237 amino acids. The folding behavior of the N-terminal domain is thus an important factor in the topogenesis of signal-anchor proteins.