Mutations in a tRNA Import Signal Define Distinct Receptors at the Two Membranes of Leishmania Mitochondria

Abstract

Nucleus-encoded tRNAs are selectively imported into the mitochondrion of Leishmania, a kinetoplastid protozoan. An oligoribonucleotide constituting the D stem-loop import signal of tRNA Tyr (GUA) was efficiently transported into the mitochondrial matrix in organello as well as in vivo. Transfer through the inner membrane could be uncoupled from that through the outer membrane and was resistant to antibody against the outer membrane receptor TAB. A number of mutations in the import signal had differential effects on outer and inner membrane transfer. Some mutants which efficiently traversed the outer membrane were unable to enter the matrix. Conversely, restoration of the loop-closing GC pair in reverse resulted in reversion of transfer through the inner, but not the outer, membrane, and binding of the RNA to the inner membrane was restored. These experiments indicate the presence at the two membranes of receptors with distinct specificities which mediate stepwise transfer into the mitochondrial matrix. The combination of oligonucleotide mutagenesis and biochemical fractionation may provide a general tool for the identification of tRNA transport factors.Mitochondria are genetic parasites presumed to have evolved from endosymbiotic bacteria. The recent sequencing of a large number of mitochondrial genomes has revealed an unexpected diversity in size and gene content (5). The loss of most of the original bacterial protein-coding genes, or their transfer to the nucleus, has been explained in terms of a "big bang" radiation of the different eukaryotic lineages from a single (monophyletic) endosymbiont (5). What is not so easily explained is the loss of a variable number of tRNA genes apparently randomly in different protists, higher plants, and at least one invertebrate lineage (6). At least 24 different tRNA species are required to read the universal genetic code. In kinetoplastid protozoa, including leishmanias and trypanosomes, a complete set of tRNAs are apparently imported in order to compensate for the total lack of mitochondrial tRNA genes (7,8,11,16,(21)(22)(23). The mitochondria of tetrahymena import about two-thirds of their tRNAs (2), while in budding yeast only a single tRNA Lys species is imported (15). In higher plants, different species mitochondrially import different sets of tRNAs; for example, mitochondria from wheat but not maize import tRNA His (9). Thus, there appears to be a speciesspecific selectivity, reflecting perhaps the presence of different mitochondrial receptors recognizing individual or groups of import signals on tRNAs.To study the basis of the selectivity of tRNA import, we have developed an in organello system using leishmania mitochondria (1,12,14). Similar systems from trypanosomes have been recently reported (18,25). Our experiments showed that tRNA import is selective, e.g., tRNATyr is efficiently imported whereas tRNA Gln (CUG) is not (1), and that a conserved sequence motif in the D arm of tRNA Tyr is necessary and sufficient for import (13). The importance of the D loop of tR...

A bifunctional tRNA import receptor from Leishmania mitochondria

“…The import signal of the type I tRNA Tyr is located within the D arm (24,25). This region contains the sequence AGAGY that is also present in the D arms of tRNA Trp and tRNA Arg , as well as in the major sequence class of type I import aptamers obtained by in vitro evolution (10), and could form the core recognition motif for RIC1͞F1␣.…”

“…Uninduced or induced cells were harvested, lysed, and separated into soluble (cytosolic) and particulate (mitochondrial) fractions (25). The crude mitochondria were treated with DNase and RNase before RNA isolation.…”

“…Unless otherwise stated, 32 Plabeled tRNA (10 fmol) was incubated with refolded recombinant protein (50 fmol) for 10 min at 4°C in 5 l of binding buffer (8) and electrophoresed on native 4-15% gradient polyacrylamide gels (12) before autoradiography. K d values of the RNAprotein complex were estimated by Scatchard analysis, as described (25). Import Assays.…”

A Sequence Motif within Trypanosome Precursor tRNAs Influences Abundance and Mitochondrial Localization

“…tRNA structure is important for mitochondrial localization in T. brucei; however, this alone may not explain the differences in the abundance of imported tRNAs (11). It has been proposed that mitochondrially localized tRNAs in Leishmania may contain sequences that positively influence targeting and transport into the mitochondrion (3,4,16,18,22). A positive import determinant was discovered in the D arms of Leishmania tRNA Ile (16) and tRNA Tyr (18).…”

“…In vitro studies with Leishmania mitochondria suggested that there might be different sequence or structural requirements for crossing the outer and inner membranes of mitochondria, indicating that import may involve two distinct steps (3). Furthermore, a SELEX (systematic evolution of ligands by exponential enrichment) procedure was used to isolate sequence aptamers that were imported into mitochondria with high efficiencies.…”