A quantitative spatial proteomics analysis of proteome turnover in human cells.

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

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-ß-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.

Dual targeting of a tRNAAsp requires two different aspartyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of the parasitic protozoon Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by partial import of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single copy genes, suggesting the use of the same enzyme in the cytosol and in the mitochondrion. However, the T. brucei genome encodes two distinct genes for eukaryotic aspartyl-tRNA synthetase (AspRS), although the cell has a single tRNAAsp isoacceptor only. Phylogenetic analysis showed that the two T. brucei AspRSs evolved from a duplication early in kinetoplastid evolution and also revealed that eight other major duplications of AspRS occurred in the eukaryotic domain. RNA interference analysis established that both Tb-AspRS1 and Tb-AspRS2 are essential for growth and required for cytosolic and mitochondrial Asp-tRNAAsp formation, respectively. In vitro charging assays demonstrated that the mitochondrial Tb-AspRS2 aminoacylates both cytosolic and mitochondrial tRNAAsp, whereas the cytosolic Tb-AspRS1 selectively recognizes cytosolic but not mitochondrial tRNAAsp. This indicates that cytosolic and mitochondrial tRNAAsp, although derived from the same nuclear gene, are physically different, most likely due to a mitochondria-specific nucleotide modification. Mitochondrial Tb-AspRS2 defines a novel group of eukaryotic AspRSs with an expanded substrate specificity that are restricted to trypanosomatids and therefore may be exploited as a novel drug target.

The single mitochondrial porin of Trypanosoma brucei is the main metabolite transporter in the outer mitochondrial membrane.

All mitochondria have integral outer membrane proteins with beta-barrel structures including the conserved metabolite transporter VDAC (voltage dependent anion channel) and the conserved protein import channel Tom40. Bioinformatic searches of the Trypanosoma brucei genome for either VDAC or Tom40 identified a single open reading frame, with sequence analysis suggesting that VDACs and Tom40s are ancestrally related and should be grouped into the same protein family: the mitochondrial porins. The single T. brucei mitochondrial porin is essential only under growth conditions that depend on oxidative phosphorylation. Mitochondria isolated from homozygous knockout cells did not produce adenosine-triphosphate (ATP) in response to added substrates, but ATP production was restored by physical disruption of the outer membrane. These results demonstrate that the mitochondrial porin identified in T. brucei is the main metabolite channel in the outer membrane and therefore the functional orthologue of VDAC. No distinct Tom40 was identified in T. brucei. In addition to mitochondrial proteins, T. brucei imports all mitochondrial tRNAs from the cytosol. Isolated mitochondria from the VDAC knockout cells import tRNA as efficiently as wild-type. Thus, unlike the scenario in plants, VDAC is not required for mitochondrial tRNA import in T. brucei.

Elongation factor 1a mediates the specificity of mitochondrial tRNA import in T. brucei.

Mitochondrial tRNA import is widespread in eukaryotes. Yet, the mechanism that determines its specificity is unknown. Previous in vivo experiments using the tRNAs(Met), tRNA(Ile) and tRNA(Lys) have suggested that the T-stem nucleotide pair 51:63 is the main localization determinant of tRNAs in Trypanosoma brucei. In the cytosol-specific initiator tRNA(Met), this nucleotide pair is identical to the main antideterminant that prevents interaction with cytosolic elongation factor (eEF1a). Here we show that ablation of cytosolic eEF1a, but not of initiation factor 2, inhibits mitochondrial import of newly synthesized tRNAs well before translation or growth is affected. tRNA(Sec) is the only other cytosol-specific tRNA in T. brucei. It has its own elongation factor and does not bind eEF1a. However, a mutant of the tRNA(Sec) expected to bind to eEF1a is imported into mitochondria. This import requires eEF1a and aminoacylation of the tRNA. Thus, for a tRNA to be imported into the mitochondrion of T. brucei, it needs to bind eEF1a, and it is this interaction that mediates the import specificity.

Conserved motifs reveal details of ancestry and structure in the small TIM chaperones of the mitochondrial intermembrane space.

The mitochondrial inner and outer membranes are composed of a variety of integral membrane proteins, assembled into the membranes posttranslationally. The small translocase of the inner mitochondrial membranes (TIMs) are a group of approximately 10 kDa proteins that function as chaperones to ferry the imported proteins across the mitochondrial intermembrane space to the outer and inner membranes. In yeast, there are 5 small TIM proteins: Tim8, Tim9, Tim10, Tim12, and Tim13, with equivalent proteins reported in humans. Using hidden Markov models, we find that many eukaryotes have proteins equivalent to the Tim8 and Tim13 and the Tim9 and Tim10 subunits. Some eukaryotes provide "snapshots" of evolution, with a single protein showing the features of both Tim8 and Tim13, suggesting that a single progenitor gene has given rise to each of the small TIMs through duplication and modification. We show that no "Tim12" family of proteins exist, but rather that variant forms of the cognate small TIMs have been recently duplicated and modified to provide new functions: the yeast Tim12 is a modified form of Tim10, whereas in humans and some protists variant forms of Tim9, Tim8, and Tim13 are found instead. Sequence motif analysis reveals acidic residues conserved in the Tim10 substrate-binding tentacles, whereas more hydrophobic residues are found in the equivalent substrate-binding region of Tim13. The substrate-binding region of Tim10 and Tim13 represent structurally independent domains: when the acidic domain from Tim10 is attached to Tim13, the Tim8-Tim13(10) complex becomes essential and the Tim9-Tim10 complex becomes dispensable. The conserved features in the Tim10 and Tim13 subunits provide distinct binding surfaces to accommodate the broad range of substrate proteins delivered to the mitochondrial inner and outer membranes.

Isolation of mitochondria from procyclic Trypanosoma brucei.

The mitochondrion of the parasitic protozoon Trypanosoma brucei shows a number of unique features, many of which represent highly interesting research topics. Studies of these subjects require the purification of mitochondrial fractions. Here, we describe and discuss the two most commonly used methods to isolate mitochondria from insect stage T. brucei. In the first protocol, the cells are lysed under hypotonic conditions, and mitoplast vesicles are isolated on Percoll gradients; in the second method, lysis occurs isotonically by N2 cavitation, and the mitochondrial vesicles are isolated by Nycodenz gradient centrifugation.

Trypanosoma seryl-tRNA synthetase is a metazoan-like enzyme with high affinity for tRNASec.

Trypanosomatids are important human pathogens that form a basal branch of eukaryotes. Their evolutionary history is still unclear as are many aspects of their molecular biology. Here we characterize essential components required for the incorporation of serine and selenocysteine into the proteome of Trypanosoma. First, the biological function of a putative Trypanosoma seryl-tRNA synthetase was characterized in vivo. Secondly, the molecular recognition by Trypanosoma seryl-tRNA synthetase of its cognate tRNAs was dissected in vitro. The cellular distribution of tRNA(Sec) was studied, and the catalytic constants of its aminoacylation were determined. These were found to be markedly different from those reported in other organisms, indicating that this reaction is particularly efficient in trypanosomatids. Our functional data were analyzed in the context of a new phylogenetic analysis of eukaryotic seryl-tRNA synthetases that includes Trypanosoma and Leishmania sequences. Our results show that trypanosomatid seryl-tRNA synthetases are functionally and evolutionarily more closely related to their metazoan homologous enzymes than to other eukaryotic enzymes. This conclusion is supported by sequence synapomorphies that clearly connect metazoan and trypanosomatid seryl-tRNA synthetases.

Dual targeting of a single tRNA(Trp) requires two different tryptophanyl-tRNA synthetases in Trypanosoma brucei.

The mitochondrion of Trypanosoma brucei does not encode any tRNAs. This deficiency is compensated for by the import of a small fraction of nearly all of its cytosolic tRNAs. Most trypanosomal aminoacyl-tRNA synthetases are encoded by single-copy genes, suggesting the use of the same enzyme in the cytosol and mitochondrion. However, the T. brucei genome contains two distinct genes for eukaryotic tryptophanyl-tRNA synthetase (TrpRS). RNA interference analysis established that both TrpRS1 and TrpRS2 are essential for growth and required for cytosolic and mitochondrial tryptophanyl-tRNA formation, respectively. Decoding the mitochondrial tryptophan codon UGA requires mitochondria-specific C-->U RNA editing in the anticodon of the imported tRNA(Trp). In vitro charging assays with recombinant TrpRS enzymes demonstrated that the edited anticodon and the mitochondria-specific thiolation of U33 in the imported tRNA(Trp) act as antideterminants for the cytosolic TrpRS1. The existence of two TrpRS enzymes, therefore, can be explained by the need for a mitochondrial synthetase with extended substrate specificity to achieve aminoacylation of the imported thiolated and edited tRNA(Trp). Thus, the notion that, in an organism, all nuclear-encoded tRNAs assigned to a given amino acid are charged by a single aminoacyl-tRNA synthetase, is not universally valid.

Mitochondrial initiation factor 2 of Trypanosoma brucei binds imported formylated elongator-type tRNA(Met).

The mitochondrion of Trypanosoma brucei lacks tRNA genes. Its translation system therefore depends on the import of nucleus-encoded tRNAs. Thus, except for the cytosol-specific initiator tRNA(Met), all trypanosomal tRNAs function in both the cytosol and the mitochondrion. The only tRNA(Met) present in T. brucei mitochondria is therefore the one which, in the cytosol, is involved in translation elongation. Mitochondrial translation initiation depends on an initiator tRNA(Met) carrying a formylated methionine. This tRNA is then recognized by initiation factor 2, which brings it to the ribosome. To guarantee mitochondrial translation initiation, T. brucei has an unusual methionyl-tRNA formyltransferase that formylates elongator tRNA(Met). In the present study, we have identified initiation factor 2 of T. brucei and shown that its carboxyl-terminal domain specifically binds formylated trypanosomal elongator tRNA(Met). Furthermore, the protein also recognizes the structurally very different Escherichia coli initiator tRNA(Met), suggesting that the main determinant recognized is the formylated methionine. In vivo studies using stable RNA interference cell lines showed that knock-down of initiation factor 2, depending on which construct was used, causes slow growth or even growth arrest. Moreover, concomitantly with ablation of the protein, a loss of oxidative phosphorylation was observed. Finally, although ablation of the methionyl-tRNA formyltransferase on its own did not impair growth, a complete growth arrest was observed when it was combined with the initiation factor 2 RNA interference cell line showing the slow growth phenotype. Thus, these experiments illustrate the importance of mitochondrial translation initiation for growth of procyclic T. brucei.