Mechanisms of Protein Sorting in Mitochondria

Stojanovski, Diana; Bohnert, Maria; Pfanner, Nikolaus; Laan, Martin van der

doi:10.1101/cshperspect.a011320

Cited by 58 publications

(62 citation statements)

References 163 publications

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“…Nuclear encoded mitochondrial proteins are directed to mitochondria by localization sequences (MLS), often found at the N or C terminus (26). These labels and additional transmembrane or localization sequences direct mitochondrial proteins to specific compartments and/or membranes (26). We chose two proteins whose MLS and specific membrane integration had been characterized by truncations fused to reporter proteins.…”

Section: Resultsmentioning

confidence: 99%

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Rosenbloom

Lee

To³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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We studied the single-molecule photo-switching properties of Dronpa, a green photo-switchable fluorescent protein and a popular marker for photoactivated localization microscopy. We found the excitation light photoactivates as well as deactivates Dronpa single molecules, hindering temporal separation and limiting super resolution. To resolve this limitation, we have developed a slowswitching Dronpa variant, rsKame, featuring a V157L amino acid substitution proximal to the chromophore. The increased steric hindrance generated by the substitution reduced the excitation light-induced photoactivation from the dark to fluorescent state. To demonstrate applicability, we paired rsKame with PAmCherry1 in a two-color photoactivated localization microscopy imaging method to observe the inner and outer mitochondrial membrane structures and selectively labeled dynamin related protein 1 (Drp1), responsible for membrane scission during mitochondrial fission. We determined the diameter and length of Drp1 helical rings encircling mitochondria during fission and showed that, whereas their lengths along mitochondria were not significantly changed, their diameters decreased significantly. These results suggest support for the twistase model of Drp1 constriction, with potential loss of subunits at the helical ends.photo-physics | PALM | suborganelle structures P hotoactivated localization microscopy (PALM) allows for subdiffraction optical resolution via the stochastic temporal separation of individual photoactivatable or reversibly photoswitchable fluorescent proteins (PA-FPs or PS-FPs) and their subsequent localization in space. Successful PALM imaging requires two conditions: (i) temporally separated stochastic activation of a population of PA-FPs or PS-FPs, and (ii) accurate localization of each molecule in space (1, 2).For PALM imaging to be more biologically applicable, robust two-color PALM is necessary. Several published protocols for two-color PALM exist, one featuring EosFP and Dronpa, a GFPlike photo-switchable protein (3-5). Initially dark, upon photoactivation by 405-nm light, Dronpa becomes fluorescent with an excitation maximum at 503 nm and an emission maximum at 515 nm. When excited by 488 nm, activated Dronpa emits green fluorescence ("ON" state) until it is photo-induced back into the dark state ("OFF" state) and can be reactivated multiple times before photobleaching (Fig. 1A). In addition, Dronpa was observed to spontaneously recover from the OFF to the ON state in tens of seconds even under 488-nm illumination alone, which hypothetically has been ascribed to thermal activation (4, 6, 7). As a result, large overlapping populations of Dronpa molecules can be excited simultaneously, especially in densely labeled samples, hindering single-molecule identification and localization. rsFastLime, a Dronpa variant, has the amino acid mutation V157G (8). The valine, not directly part of the chromophore, is involved in the photoactivated isomerization of the chromophore. The V157G mutation is thought to reduce the steric ...

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Section: Resultsmentioning

confidence: 99%

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Rosenbloom

Lee

To³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…In view of the general lack of substrate specificity observed in this work, all proteins transiting the IMS en route to their final location are potential substrates of the Mia40/lfALR system. However oxidation might be prevented by efficient sequestration during interactions with translocases [12, 13, 59] or chaperones [59, 60], or might be reversed via IMS-resident reductase sytems [23, 61–63]. In terms of an expanding role for the Mia40/lfALR system, it is interesting to note that a mitochondrial matrix protein Mrp10 contains two disulfide bonds that are inserted during transit through the IMS [34].…”

Section: Resultsmentioning

confidence: 99%

Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity

Hudson

Thorpe

2015

Archives of Biochemistry and Biophysics

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Mia40 participates in oxidative protein folding within the mitochondrial intermembrane space (IMS) by mediating the transfer of reducing equivalents from client proteins to FAD-linked oxidoreductases of the Erv1 family (lfALR in mammals). Here we investigate the specificity of the human Mia40/lfALR system towards non-cognate unfolded protein substrates to assess whether the efficient introduction of disulfides requires a particular amino acid sequence context or the presence of an IMS targeting signal. Reduced pancreatic ribonuclease A (rRNase), avian lysozyme, and riboflavin binding protein are all competent substrates of the Mia40/lfALR system, although they lack those sequence features previously thought to direct disulfide bond formation in cognate IMS substrates. The oxidation of rRNase by Mia40 does not limit overall turnover of unfolded substrate by the Mia40/lfALR system. Mia40 is an ineffective protein disulfide isomerase when its ability to restore enzymatic activity from scrambled RNase is compared to that of protein disulfide isomerase. Mia40’s ability to bind amphipathic peptides is evident by avid binding to the isolated B-chain during the insulin reductase assay. In aggregate these data suggest that the Mia40/lfALR system has a broad sequence specificity and that potential substrates may be protected from adventitious oxidation by kinetic sequestration within the mitochondrial IMS.

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“…Together, these data argue that TASK-3 channels can be sorted to mitochondria in steroidogenic cells, extending findings of an earlier report documenting TASK-3 channel expression in mitochondria of melanoma malignum cells 38 . Although TASK-3 is missing a discrete amino terminal mitochondrial targeting sequence, many proteins that lack such a signal are imported to mitochondria by internal targeting sequences or cytosolic chaperone proteins 39 .…”

Section: Discussionmentioning

confidence: 99%

Functional TASK-3–Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells

et al. 2017

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Ca2+ drives aldosterone synthesis in the cytosolic and mitochondrial compartments of the adrenal zona glomerulosa (ZG) cell. Membrane potential across each of these compartments regulates the amplitude of the Ca2+ signal; yet, only plasma membrane ion channels and their role in regulating cell membrane potential have garnered investigative attention as pathological causes of human hyperaldosteronism. Previously, we reported that genetic deletion of TASK-3 channels from mice produces aldosterone excess in the absence of a change in the cell membrane potential of ZG cells. Here, we report using yeast two-hybrid, immunoprecipitation and electron microscopic analyses that TASK-3 channels are resident in mitochondria, where they regulate mitochondrial morphology, mitochondrial membrane potential (mitoVm) and aldosterone production. This study provides proof-of-principle that mitochondrial K+ channels, by modulating inner mitochondrial membrane morphology and mitoVm, have the ability to play a pathological role in aldosterone dysregulation in steroidogenic cells.

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Mechanisms of Protein Sorting in Mitochondria

Cited by 58 publications

References 163 publications

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Optimized two-color super resolution imaging of Drp1 during mitochondrial fission with a slow-switching Dronpa variant

Mia40 is a facile oxidant of unfolded reduced proteins but shows minimal isomerase activity

Functional TASK-3–Like Channels in Mitochondria of Aldosterone-Producing Zona Glomerulosa Cells

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