Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex. The complex contains three mitochondrion- and eight nuclear-encoded subunits; six of the latter are necessary and sufficient for import. Antisense-mediated knockdown of essential subunits resulted in the depletion of mitochondrial tRNAs and inhibition of organellar translation. Functional complexes were reconstituted with recombinant subunits expressed in Escherichia coli. Several essential RIC subunits are identical to specific subunits of respiratory complexes. These findings provide new information on the evolution of tRNA import and the foundation for detailed structural and mechanistic studies.     

The direct route: a simplified pathway for protein import into the mitochondrion of trypanosomes

Trypanosoma brucei is a unicellular eukaryote that causes the deadly human African trypanosomiasis ('sleeping sickness') in humans. The parasite has a complicated lifestyle, it developmentally changes aspects of its mitochondrial function as it alternates from forms in the tsetse fly to forms adapted for life in the human bloodstream. The single mitochondrion found in each trypanosome has to be duplicated precisely in each round of the cell cycle in order for parasites to replicate, and this depends on the import of proteins from the cytosol. Here we review what is known about the mitochondrial protein import pathway in T. brucei, how it compares with the process in humans, and how the distinguishing features seen in T. brucei and humans promise new understanding of the mitochondrial protein import process in all eukaryotes.     

Necessary and sufficient conditions for evolutionary suicide

Evolutionary suicide is an evolutionary process where a viable population adapts in such a way that it can no longer persist. It has already been found that a discontinuous transition to extinction is a necessary condition for suicide. Here we present necessary and sufficient conditions, concerning the bifurcation point, for suicide to occur. Evolutionary suicide has been found in structured metapopulation models. Here we show that suicide can occur also in unstructured population models. Moreover, a structured model does not guarantee the possibility of suicide: we show that suicide cannot occur in age-structured population models of the Gurtin-MacCamy type. The point is that the mutant’s fitness must explicitly depend not only on the environmental interaction variable, but also on the resident strategy.     

The C-terminal globular domain of the prion protein is necessary and sufficient for import into the endoplasmic reticulum

The mammalian prion protein (PrP) is composed of an unstructured flexible N-terminal region and a C-terminal globular domain. We examined the import of PrP into the endoplasmic reticulum (ER) of neuronal cells and show that information present in the C-terminal globular domain is required for ER import of the N terminus. N-terminal fragments of PrP, devoid of structural domains located in the C terminus, remained in the cytosol with an uncleaved signal peptide and were rapidly degraded by the proteasome. Conversely, the separate C-terminal domain of PrP, comprising the highly ordered helix 2-loop-helix 3 motif, was entirely imported into the ER. As a consequence, two PrP mutants linked to inherited prion disease in humans, PrP-W145Stop and PrP-Q160Stop, were partially retained in the cytosol. The cytosolic fraction was characterized by an uncleaved N-terminal signal peptide and was degraded by the proteasome. Our study identified a new regulatory element in the C-terminal globular domain of PrP necessary and sufficient to promote import of PrP into the ER.     

Ancestral and derived protein import pathways in the mitochondrion of Reclinomonas americana

The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or "translocase in the inner mitochondrial membrane complex." Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.     

Protein import, replication, and inheritance of a vestigial mitochondrion

Mitochondrial remnant organelles (mitosomes) that exist in a range of "amitochondrial" eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often described as the earliest branching eukaryote, contains double membrane-bounded structures involved in iron-sulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways akin to those that operate in mammalian mitochondria. Although import of IscU and ferredoxin is still reliant on their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer requires cleavable presequences, a derived feature from their mitochondrial homologues. In addition, we found that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle, whereas peripherally located mitosomes are inherited into daughter cells stochastically.     

In vivo study in Trypanosoma brucei links mitochondrial transfer RNA import to mitochondrial protein import

Trypanosoma brucei imports all mitochondrial transfer RNAs (tRNAs) from the cytosol. By using cell lines that allow independent tetracycline-inducible RNA interference and isopropyl-β-D-thiogalactopyranoside-inducible expression of a tagged tRNA, we show that ablation of Tim17 and mitochondrial heat-shock protein 70, components of the inner-membrane protein translocation machinery, strongly inhibits import of newly synthesized tRNAs. These findings, together with previous results in yeast and plants, suggest that the requirement for mitochondrial protein-import factors might be a conserved feature of mitochondrial tRNA import in all systems.     

Biosynthesis and import into the mitochondrion of L-3-glycerophosphate dehydrogenase, and the effect of thyroid hormone deficiency on gene expression

Mitochondrial L-3-glycerophosphate dehydrogenase (EC 1.1.99.5) is synthesised in bovine kidney (NBL-1) cells treated with uncoupler as a cytosolic precursor with Mr = 76,000 indistinguishable from the mature form. In vitro translation of rat liver mRNA also gives rise to a product of Mr = 76,000 but when this is imported into mitochondria it is processed to a product of Mr = 66,000. L-3-Glycerophosphate dehydrogenase activity and immunoreactive protein are greatly decreased in liver mitochondria from hypothyroid rats. Paradoxically, in vitro translation of the mRNA from such animals gives rise to large amounts of the protein, much greater than that synthesised from euthyroid mRNA and comparable with that produced from hyperthyroid mRNA.     

Necessary and sufficient conditions for the existence of an optimisation principle in evolution

This paper presents a necessary condition for the existence of a numerical quantity optimised by evolution by natural selection, which also turns out to be a sufficient condition under rather general conditions. As a corollary, a related criterion with a particularly intuitive graphical interpretation in terms of pairwise invadability plots is obtained.     

Necessary and sufficient conditions for the existence of linear invariants in phylogenetic inference.

The necessary and sufficient conditions for the existence of linear invariants under semigroups of probability transition matrices are derived. It is found that a biologically meaningful nucleotide substitution model has linear invariants if and only if it is a submodel of one of the three most general models, which include the so-called balanced and unbalanced transversion models. Each of these three general models is a nucleotide substitution model with six parameters.     

Kinetin incorporated into tobacco callus ribosomal RNA and transfer RNA preparations

Kinetin, N⁶-furfuryladenine, was incorporated into tobacco (nicotiana tabacum L., var. Wis. No. 38) callus RNA isolated from rapidly growing tissue cultured in the presence of N⁶-furfuryladenine-8-¹⁴C or unlabeled kinetin. Approximately 0.7% of the radioactivity in the labeled kinetin added to the medium was recovered as N⁶-furfuryladenosine (fr⁶A) in the rRNA and tRNA preparations from the tobacco callus. The rRNA contained over 90% of these fr⁶ A moieties. The extent of kinetin incorporation was four times greater than that observed for N⁶-benzyladenine. The radiochemical purity of the recovered fr⁶ A was confirmed by three successive chromatographic purifications on Sephadex columns (LH-20 eluted with 35% ethanol, G-10 eluted with 20% ethanol, and LH-20 eluted with water). A cytokinin-active ribonucleoside with elution volumes corresponding to fr⁶ A was isolated from the tobacco callus rRNA preparation. This compound was analyzed by gas-liquid chromatography and rigorously characterized as N⁶-furfuryladenosine by gas-liquid chromatography-mass spectrometry of the trimethylsilyl derivative.     

Queuine is incorporated into brain transfer RNA

Pig brain tRNA was assayed for the presence of queuosine in the first position of the anticodon for each of the Q-family of tRNAs (aspartyl, asparaginyl, histidyl and tyrosyl). The brain tRNA was aminoacylated with each of the four amino acids and the aminoacylated tRNA's analyzed by RPC-5 chromatography. The results of this study show that for all four tRNAs of the family, queuine is substituted for guanine in virtually 100% of the anticodons. Therefore, it can be concluded that queuine is able to cross the blood-brain barrier and that brain contains quanine-queuine tRNA transglycosylase, the enzyme responsible for the excision of guanine from the orginal transcipts of these tRNAs and insertion of queuine. The determination of whether the tRNA contained queuine was made from the elution profile of the RPC-5 chromatrograms and the results confirmed by a change in the RPC-5 elution profile when the tRNAs were reacted with BrCN or NaIO₄.     

Recycling factors for ribosome disassembly in the apicoplast and mitochondrion of Plasmodium falciparum

The reduced genomes of the apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are actively translated and antibiotic‐mediated translation inhibition is detrimental to parasite survival. In order to understand recycling of organellar ribosomes, a critical step in protein translation, we identified ribosome recycling factors (RRF) encoded by the parasite nuclear genome. Targeting of PfRRF1 and PfRRF2 to the apicoplast and mitochondrion respectively was established by localization of leader sequence–GFP fusions. Unlike any RRF characterized thus far, PfRRF2 formed dimers with disulphide interaction(s) and additionally localized in the cytoplasm, thus suggesting adjunct functions for the factor. PfRRF1 carries a large 108‐amino‐acid insertion in the functionally critical hinge region between the head and tail domains of the protein, yet complemented Escherichia coli RRF in the LJ14frr^ts mutant and disassembled surrogate E. coli 70S ribosomes in the presence of apicoplast‐targeted EF‐G. Recombinant PfRRF2 bound E. coli ribosomes and could split monosomes in the presence of the relevant mitochondrial EF‐G but failed to complement the LJ14frr^ts mutant. Although proteins comprising subunits of P. falciparum organellar ribosomes are predicted to differ from bacterial and mitoribosomal counterparts, our results indicate that the essential interactions required for recycling are conserved in parasite organelles.     

Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Non-canonical transit peptide for import into the chloroplast

The large majority of plastid proteins are nuclear-encoded and, thus, must be imported within these organelles. Unlike most of the outer envelope proteins, targeting of proteins to all other plastid compartments (inner envelope membrane, stroma, and thylakoid) is strictly dependent on the presence of a cleavable transit sequence in the precursor N-terminal region. In this paper, we describe the identification of a new envelope protein component (ceQORH) and demonstrate that its subcellular localization is limited to the inner membrane of the chloroplast envelope. Immunopurification, microsequencing of the natural envelope protein and cloning of the corresponding full-length cDNA demonstrated that this protein is not processed in the N-terminal region during its targeting to the inner envelope membrane. Transient expression experiments in plant cells were performed with truncated forms of the ceQORH protein fused to the green fluorescent protein. These experiments suggest that neither the N-terminal nor the C-terminal are essential for chloroplastic localization of the ceQORH protein. These observations are discussed in the frame of the endosymbiotic theory of chloroplast evolution and suggest that a domain of the ceQORH bacterial ancestor may have evolved so as to exclude the general requirement of an N-terminal plastid transit sequence.