TimX, a novel player in protein import across the inner mitochondrial membrane of T. brucei

Mitochondrial protein import is an essential function of the unique mitochondrion in T. brucei as roughly 1000 different nuclear encoded proteins need to be correctly localized to their mitochondrial subcompartment. For this reason the responsible import machinery is expected to be similarly complex as in other Eukaryotes. This was recently demonstrated for the translocation machinery in the outer mitochondrial membrane. In contrast, the composition of the inner membrane import machinery and the exact molecular pathway(s) taken by various substrates are still ill-defined. To elucidate this further, we performed a pulldown analysis of epitope tagged TbTim17 in combination with quantitative mass spectrometry. By this we identified novel components of the mitochondrial import machinery in trypanosomes. One of these, TimX, is an essential mitochondrial membrane protein of 42 kDa that is unique to kinetoplastids. This protein migrates on Blue Native PAGE in a high molecular weight complex similar to TbTim17. Ablation of either of the two proteins leads to a destabilization of the complex containing the other protein. Furthermore, its involvement in protein import could be demonstrated by in vivo and in vitro protein import assays. This corroborates that TimX together with TbTim17 forms a protein import complex in the inner mitochondrial membrane. As TbTim17 the TimX protein was subjected to pulldown analysis in combination with quantitative mass spectrometry. The overlap of candidates defined by these two sets of IPs likely defines further components of the inner membrane translocase which are presently being analyzed. In summary our study on novel components of the trypanosome mitochondrial protein import system gives us fascinating new insights into evolution of the mitochondrion.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Their protein import systems are of similar complexity yet different composition than those in other eukaryotes. To elucidate the composition of the trypanosomal translocase of the inner mitochondrial membrane (TIM) we performed CoIPs of epitope-tagged TbTim17 and two other candidates in combination with SILAC-based quantitative mass spectrometry. This led to the identification of ten candidates for core TIM subunits. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a trypanosomatid-specific 42 kDa protein (Tim42) and two putative orthologues of inactive rhomboid proteases were analyzed further. All three proteins are essential in both life cycle stages and their ablation results in a strong protein import defect in vivo and in vitro. Blue native PAGE revealed their presence in a high molecular weight complex. Unlike anticipated, trypanosomes have a highly complex TIM translocase that has extensively been redesigned. None of the three novel TIM subunits has ever been associated with mitochondrial protein import. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. This suggests an exciting analogy between protein translocases of mitochondria and the ER.

The TIM translocase in T. brucei contains Rhomboid-like proteins that are essential for mitochondrial protein import

The parasitic protozoon Trypanosoma brucei is often considered as one of the earliest branching eukaryotes that have mitochondria capable of oxidative phosphorylation. Its protein import systems are therefore of great interest. Recently, it was shown that the outer mitochondrial membrane protein translocase is of similar complexity yet different composition than in other eukaryotes (1). In the inner membrane however, only a single orthologue of the pore forming Tim17/22/23 protein family was identified and termed TbTim17. Based on this finding it has been suggested that, instead of separate TIM22 and TIM23 complexes as in other eukaryotes, trypanosomes may have a single multifunctional translocase of the inner mitochondrial membrane (TIM) of reduced complexity. To elucidate the composition of the trypanosomal TIM complex we performed co-immunoprecipitations (CoIP) of epitope-tagged TbTim17 in combination with SILAC-based quantitative mass spectrometry. This led to the identification of 22 highly enriched TbTim17-interacting proteins. We tagged two of the top-scoring proteins for reciprocal CoIP analyses and recovered a set of ten proteins that are highly enriched in all three CoIPs. These proteins are excellent candidates for core subunits of the trypanosomal TIM complex. Eight of them were present in the previously determined inner membrane proteome and four show homology to small Tim chaperones. Three candidates, a novel trypanosomatid-specific 42 kDa protein, termed Tim42, and two putative orthologues of probably inactive rhomboid proteases were chosen for further analysis. All three proteins are essential in both life cycle stages and in a cell line that can grow in the absence of mitochondrial DNA. Additionally, their ablation by RNAi results in a strong protein import defect both in vivo and in vitro. Blue native PAGE reveals that Tim42, like TbTim17 is present in a high molecular weight complex. Moreover, ablation of either Tim42 or TbTim17 leads to a destabilization of the complex containing the other protein, suggesting a tight interaction of the two proteins. In summary our study shows that unlike anticipated trypanosomes have a highly complex TIM translocase that has extensively been redesigned. We have characterized three novel TIM subunits that have never been associated with mitochondrial protein import before. Two of them belong to the rhomboid protease family, a member of which recently has been implicated in the ERAD translocation system. Our study provides insight into mitochondrial evolution over large phylogenetic distances and suggests an exciting analogy between protein translocation systems of mitochondria and the ER.

pATOM36 mediates mitochondrial protein import and mitochondrial DNA inheritance

The multisubunit ATOM complex mediates import of essentially all proteins across the outer mitochondrial membrane in T. brucei. Moreover, an additional protein termed pATOM36, which is loosely associated with the ATOM complex, has been implicated in the import of only a subset of mitochondrial matrix proteins. Here we have investigated more precisely which role pATOM36 plays in mitochondrial protein import. RNAi mediated ablation of pATOM36 specifically depletes a subset of ATOM complex subunits and as a consequence results in the collapse of the ATOM complex as shown by Blue native PAGE. In addition, a SILAC-based global proteomic analysis of uninduced and induced pATOM36 RNAi cells together with in vitro import experiments suggest that pATOM36 might be a novel protein insertase acting on a subset of alpha-helically anchored mitochondrial outer membrane proteins. Identification of pATOM36 interaction partners by co-immunoprecipitation together with immunofluorescence analysis furthermore shows that unexpectedly a fraction of the protein is associated with the tripartite attachment complex (TAC). This complex is essential for proper inheritance of the mtDNA; also called kinetoplast or kDNA; as it forms a physical connection between the kDNA and the basal body of the single flagellum throughout the cell cycle. Thus, the presence of pATOM36 in the TAC provides an exciting link between mitochondrial protein import and kDNA inheritance.

Assembly, nuclear import and function of U7 snRNPs studied by microinjection of synthetic U7 RNA into Xenopus oocytes.

In Xenopus oocytes in vitro transcribed mouse U7 RNA is assembled into small nuclear ribonucleoproteins (snRNPs) that are functional in histone RNA 3' processing. If the special Sm binding site of U7 (AAUUUGUCUAG, U7 Sm WT) is converted into the canonical Sm sequence derived from the major snRNAs (AAUUUUUGGAG, U7 Sm OPT) the RNA assembles into a particle which accumulates more efficiently in the nucleus, but which is non-functional. U7 RNA with a heavily mutated Sm binding site (AACGCGUCAUG, U7 Sm MUT) is deficient in nuclear accumulation and function. By UV cross-linking U7 Sm WT RNA can be linked to three proteins, i.e. the common snRNP proteins G and B/B' and an apparently U7-specific protein of 40 kDa. As a result of altering the Sm binding site, U7 Sm OPT RNA cannot be cross-linked to the 40 kDa protein and no cross-links are obtained with U7 Sm MUT RNA. The fact that the Sm site also interacts with at least one U7-specific protein is so far unique to U7 RNA and may provide an explanation for the atypical sequence of this site. All described RNA-protein interactions, including that with the 40 kDa protein, already occur in the cytoplasm. An additional cytoplasmic photoadduct obtained with U7 Sm WT and U7 Sm OPT, but not U7 Sm MUT, RNAs is indicative of a protein of 60-80 kDa. The m7G cap structure of U7 Sm WT and U7 Sm OPT RNA becomes hypermethylated. However, the 3mG cap enhances, but is not required for, nuclear accumulation. Finally, U7 Sm WT RNA is functional in histone RNA processing even when bearing an ApppG cap.

Costly Revenue-Raising and the Case for Favoring Import-Competing Industries

A standard finding in the political economy of trade policy literature is that we should expect export-oriented industries to attract more assistance than import-competing industries. In reality, however, trade policy is heavily biased toward supporting import industries. This paper shows within a standard protection for sale framework, how the costliness of raising revenue via taxation makes trade subsidies less desirable and trade taxes more desirable. The model is then estimated and its predictions tested using U.S. tariff data. An empirical estimate of the costliness of revenue-raising is also obtained.

Physa acuta, die Spitz-Quellschnecke, ein unbeabsichtigter Import in unseren Aquarien

Von Walter Köhler

Multibeam Converter - a tool to create Ocean Data View import files from multibeam files

The software Multibeam Converter is a tool to convert files or folders of files (ascii/tab-separated data files with or without metaheader), downloaded from PANGAEA via the search engine or the data warehouse to the ODV import format, e.g. for visualization or further processing. MultibeamConverter is distributed as freeware for the operating systems Microsoft Windows, Apple OS X and Linux.

Split2Events - a tool for preparing PANGAEA import files

Split2Events is a software tool to split one file with data from several events into several files, one for each event. The resulting folder with a number of files can automaticaly be imported with the Massenimport routine of 4D. But it can also be useful to configure a complex import file outside 4D. Split2Events may also extract a list of unknown parameters and create a parameter import file.

PanConverter - a tool for data conversion and recalculation, written to convert special data collections to standard import format used by PANGAEA

Supported file formats: - CrossRef XML file(s) - TRiDaS (Tree Ring Data Standard, http://www.tridas.org). Example: hdl:10013/epic.42747.d001 - IMMA (International Maritime Meteorological Archive). Used by the project CLIWOC (García-Herrera et al. 2007, http://doi.pangaea.de/10.1594/PANGAEA.743343) - NOAA IOAS (International Ocean Atlas Series). Example: hdl:10013/epic.42747.d008 - SOCAT (Surface Ocean CO2 Atlas, Bakker et al. 2014, http://doi.pangaea.de/10.1594/PANGAEA.811776) - CHUAN (Comprehensive Historical Upper-Air Network, Stickler et al. 2013, http://doi.pangaea.de/10.1594/PANGAEA.821222). Example: hdl:10013/epic.42747.d003 - Thermosalinograph (TSG) data. Format developed by Gerd Rohardt. Example: hdl:10013/epic.42747.d002 - Columus GPS Data Logger V-900 format to KML or GPX. Example: hdl:10013/epic.42747.d006

The Role of Distance in Determining International Transport Costs: Evidence from Philippine Import Data

This paper presents an empirical investigation of the appropriateness of distance as a determinant of international transport costs by using Philippine import data. This study addresses three specific questions. First, does distance really matter in the determination of transport costs? Second, if distance is a significant factor, what is the magnitude of its impact? Third, does the impact of distance on transport costs vary by commodity? Results indicate that while distance is important in determining transport costs, using distance alone as the proxy of international transport costs is insufficient, and such use underestimates the impact of distance on international transport costs. Results also indicate that the impact of distance varies across commodity groups, but it is difficult to precisely determine the direction and the magnitude of this impact.

Smuggling and import duties in Myanmar

This paper examines the effects of import duties on smuggling in Myanmar. Following Fisman and Wei (2004), the reporting discrepancies between Myanmar’s imports records and corresponding exports recorded by trading partners are regarded as indicative of smuggling. The paper studies whether reporting discrepancies differ across trading partners as well as across time. Our main findings are first, that the hike in import duties in June 2004 helped to widen the reporting discrepancies, which suggests smuggling for tax evasion purposes and second, that reporting discrepancies differ considerably across trading partners: land borders appear to be particularly attractive venues for smugglers.

How do free trade agreements change import prices? : firm-level evidence from China's imports from ASEAN

The literature has revealed the positive impacts of free trade agreements (FTAs) on export prices by employing product-level trade data. This paper empirically examines the impacts of FTAs on import prices at the firm level. We focus on firm-level imports in China from ASEAN countries by employing China’s firm-product-level trade data. As a result, controlling for firm characteristics and product characteristics, we could not find significantly positive impacts of an FTA’s entry into force on import prices of FTA eligible products. Instead, we found a significant increase in import quantities of FTA eligible products. Thus, at the firm level, the gains from FTAs for exporters may be the increase in export quantities rather than the rise in export prices.

Why do oil importers diversify their import sources politically? : evidence from U.S. firm-level data

International politics affects oil trade. But why? We construct a firm-level dataset for all U.S. oil-importing companies over 1986-2008 to examine what kinds of firms are more responsive to change in "political distance" between the U.S. and her trading partners, measured by divergence in their UN General Assembly voting patterns. Consistent with previous macro evidence, we first show that individual firms diversify their oil imports politically, even after controlling for unobserved firm heterogeneity. We conjecture that the political pattern of oil imports from these individual firms is driven by hold-up risks, because oil trade is often associated with backward vertical FDI. To test this hold-up risk hypothesis, we investigate heterogeneity in responses by matching transaction-level import data with firm-level worldwide reserves. Our results show that long-run oil import decisions are indeed more elastic for firms with oil reserves overseas than those without, although the reverse is true in the short run. We interpret this empirical regularity as that while firms trade in the spot market can adjust their imports immediately, vertically-integrated firms with investment overseas tend to commit to term contracts in the short run even though they are more responsive to changes in international politics in the long run.