The mitochondrial uniporter is a highly selective calcium channel in the organelle’s inner membrane. Its molecular components include the EF-hand containing proteins mitochondrial calcium uptake 1 (MICU1) and MICU2 and the pore forming subunit mitochondrial calcium uniporter (MCU). We sought to achieve a full molecular characterization of the uniporter holocomplex (uniplex). Quantitative mass spectrometry of affinity-purified uniplex recovered MICU1 and MICU2, MCU and its paralog MCUb, and essential MCU regulator (EMRE), a previously uncharacterized protein. EMRE is a 10 kD, metazoan specific protein with a single transmembrane domain. In its absence, uniporter channel activity was lost despite intact MCU expression and oligomerization. EMRE was required for the interaction of MCU with MICU1 and MICU2. Hence, EMRE is essential for in vivo uniporter current and additionally bridges the calcium-sensing role of MICU1 and MICU2 with the calcium conducting role of MCU.
The mitochondrial calcium uniporter is a highly selective calcium channel distributed broadly across eukaryotes but absent in the yeast Saccharomyces cerevisiae. The molecular components of the human uniporter holocomplex (uniplex) have been identified recently. The uniplex consists of three membrane-spanning subunits -mitochondrial calcium uniporter (MCU), its paralog MCUb, and essential MCU regulator (EMRE)-and two soluble regulatory components-MICU1 and its paralog MICU2. The minimal components sufficient for in vivo uniporter activity are unknown. Here we consider Dictyostelium discoideum (Dd), a member of the Amoebazoa outgroup of Metazoa and Fungi, and show that it has a highly simplified uniporter machinery. We show that D. discoideum mitochondria exhibit membrane potential-dependent calcium uptake compatible with uniporter activity, and also that expression of DdMCU complements the mitochondrial calcium uptake defect in human cells lacking MCU or EMRE. Moreover, expression of DdMCU in yeast alone is sufficient to reconstitute mitochondrial calcium uniporter activity. Having established yeast as an in vivo reconstitution system, we then reconstituted the human uniporter. We show that coexpression of MCU and EMRE is sufficient for uniporter activity, whereas expression of MCU alone is insufficient. Our work establishes yeast as a powerful in vivo reconstitution system for the uniporter. Using this system, we confirm that MCU is the pore-forming subunit, define the minimal genetic elements sufficient for metazoan and nonmetazoan uniporter activity, and provide valuable insight into the evolution of the uniporter machinery.
Tic110 has been proposed to be a channel-forming protein at the inner envelope of chloroplasts whose function is essential for the import of proteins synthesized in the cytosol. Sequence features and topology determination experiments presently summarized suggest that Tic110 consists of six transmembrane helices. Its topology has been mapped by limited proteolysis experiments in combination with mass spectrometric determinations and cysteine modification analysis. Two hydrophobic transmembrane helices located in the N terminus serve as a signal for the localization of the protein to the membrane as shown previously. The other amphipathic transmembrane helices are located in the region composed of residues 92-959 in the pea sequence. This results in two regions in the intermembrane space localized to form supercomplexes with the TOC machinery and to receive the transit peptide of preproteins. A large region also resides in the stroma for interaction with proteins such as molecular chaperones. In addition to characterizing the topology of Tic110, we show that Ca 2؉ has a dramatic effect on channel activity in vitro and that the protein has a redox-active disulfide with the potential to interact with stromal thioredoxin.After the proposed endosymbiotic event that gave rise to chloroplasts and, as a consequence, to the massive transfer of genes from the endosymbiont to the host cell nucleus, two new machineries were developed at the outer and inner envelope of chloroplasts, the TOC and TIC complexes, respectively. The machineries transport plastid proteins, whose synthesis was moved to the cytosol, back to the plastid (for a review, see Refs. 1 and 2). Whereas the channel of the TOC complex is homologous to the Omp85 family of transporters in bacteria (3), the TIC complex shows no homology to known transport systems, since the plasma membrane-bacterial transport machineries were relocated to the thylakoid membrane. Moreover, there was an essential requirement for establishing new lines of communication between the organelle and other parts of the cell (4). In chloroplasts of land plants, redox regulation is linked to oxygen and/or light. The regulation of chloroplast-specific processes by redox in accord with the status of the organelle applies not only for transcription and translation but also for translocation. Redox regulation at the translocation level has been proposed for the TIC machinery on the basis of protein composition and dynamics (5-8) as well as specific in vitro import experiments (9). However, despite advances in our biochemical understanding of the import process, little is presently known regarding details of the role of redox or of the structure of the TIC complex, particularly with respect to its channel subunits.Tic110 is essential for protein import into plastids (10 -12). It has been proposed to be a pore-forming protein and one of the main components of the TIC complex (13). Previous studies have provided information about certain aspects of Tic110 structure-function. The region from residu...
BackgroundThe Tic complex (Translocon at the inner envelope membrane of chloroplasts) mediates the translocation of nuclear encoded chloroplast proteins across the inner envelope membrane. Tic110 forms one prominent protein translocation channel. Additionally, Tic20, another subunit of the complex, was proposed to form a protein import channel - either together with or independent of Tic110. However, no experimental evidence for Tic20 channel activity has been provided so far.ResultsWe performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20-proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro.ConclusionsThe presented data provide first biochemical evidence to the notion that Tic20 can act as a channel protein within the chloroplast import translocon complex. However, the very low abundance of Tic20 in the inner envelope membranes indicates that it cannot form a major protein translocation channel. Furthermore, the independent complex formation of Tic20 and Tic110 argues against a joint channel formation. Thus, based on the observed channel activity of Tic20 in proteoliposomes, we speculate that the chloroplast inner envelope contains multiple (at least two) translocation channels: Tic110 as the general translocation pore, whereas Tic20 could be responsible for translocation of a special subset of proteins.
Chloroplasts like mitochondria were derived from an endosymbiontic event. Due to the massive gene transfer to the nucleus during endosymbiosis, only a limited number of chloroplastic proteins are still encoded for in the plastid genome. Most of the nuclear-encoded plastidic proteins are post-translationally translocated back to the chloroplast via the general import pathway through distinct outer and inner envelope membrane protein complexes, the Toc and Tic translocons (Translocon at the outer/inner envelope membrane of chloroplasts). Eight Tic subunits have been described so far, including two potential channel proteins (Tic110 and Tic20), the "motor complex" (Tic40 associated with the stromal chaperone Hsp93) and the "redox regulon" (Tic62, Tic55, and Tic32) involved in regulation of protein import via the metabolic redox status of the chloroplast. Regulation can additionally occur via thioredoxins (Tic110 and Tic55) or via the calcium/calmodulin network (Tic110 and Tic32). In this review we present the current knowledge about the Tic complex focusing on its regulation and addressing some still open questions.
Electrical signals in excitable tissues such as nerves and muscles are generated by the synchronized opening and closing of ion channels in cell membranes, which generate transient transmembrane gradients in the concentrations of sodium and potassium ions. These channels open and close in response to local
In contrast to the damaging effect of high-concentration chemical stressors, the same agents in very low (submicromolar) concentrations have a positive effect on the treated plants, which is non-specific (independent of the chemical nature of the agent). The direct responses depend on the treated organ. When leaves are treated, the effects include an increase in chlorophyll content, CO(2) fixation and delaying senescence of chloroplasts. When roots are treated, the direct effect is an increased cytokinin synthesis. This hormone, after being transported to the shoot, exerts secondary effects, which are similar to the primary ones in leaves. The signalization routes involved in the primary effects proved to be the phosphoinositide and MAPK pathways in any stimulated organ. In this mini-review we summarize our current knowledge about the effects of low-concentration stressors and their mechanism of action with the help of the four used model systems: detached non-rooting and rooting leaves, hydroponically treated and sprayed seedlings.
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