The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

Wenz, Lena‐Sophie; Opaliński, Łukasz; Schuler, Max‐Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; Laan, Martin van der; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

doi:10.1002/embr.201338144

Cited by 43 publications

(64 citation statements)

References 34 publications

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“…One possibility is that the reduced PC levels affect the function of the TOM complex. However, the accumulation of TIM23-dependent precursors like Om45-DHFR (81,82) and Oxa1 at the TOM complex was not decreased (Fig. 6B) (75).…”

Section: Depletion Of Pc Impairs Protein Transport Into the Innermentioning

confidence: 92%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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Two protein translocases transport precursor proteins into or across the inner mitochondrial membrane. The presequence translocase (TIM23 complex) sorts precursor proteins with a cleavable presequence either into the matrix or into the inner membrane. The carrier translocase (TIM22 complex) inserts multispanning proteins into the inner membrane. Both protein import pathways depend on the presence of a membrane potential, which is generated by the activity of the respiratory chain. The non-bilayer-forming phospholipids cardiolipin and phosphatidylethanolamine are required for the activity of the respiratory chain and therefore to maintain the membrane potential for protein import. Depletion of cardiolipin further affects the stability of the TIM23 complex. The role of bilayer-forming phospholipids like phosphatidylcholine (PC) in protein transport into the inner membrane and the matrix is unknown. Here, we report that import of presequence-containing precursors and carrier proteins is impaired in PC-deficient mitochondria. Surprisingly, depletion of PC does not affect stability and activity of respiratory supercomplexes, and the membrane potential is maintained. Instead, the dynamic TIM23 complex is destabilized when the PC levels are reduced, whereas the TIM22 complex remains intact. Our analysis further revealed that initial precursor binding to the TIM23 complex is impaired in PC-deficient mitochondria. We conclude that reduced PC levels differentially affect the TIM22 and TIM23 complexes in mitochondrial protein transport.Mitochondria fulfill essential functions for the survival of the cell like energy conversion to produce ATP, synthesis of amino acids, lipids, and heme, as well as the generation of iron-sulfur clusters. They contain about 1000 proteins in yeast and 1500 proteins in humans (1, 2). More than 99% of the mitochondrial proteins are synthesized as precursors on cytosolic ribosomes. Mitochondria contain a sophisticated system of protein translocases to import precursor proteins (3-9). The translocase of the outer membrane (TOM 3 complex) forms the general entry gate for most precursor proteins. After passage of the TOM channel, distinct protein translocases sort the preproteins into the different subcompartments: the outer and inner membrane as well as the two aqueous compartments, the matrix and intermembrane space.The majority of mitochondrial proteins are sorted into the inner membrane and the matrix. Two inner membrane-bound protein complexes mediate protein import. The presequence translocase (also termed TIM23 complex) transports precursor proteins with a cleavable presequence into the inner membrane and the matrix, whereas the carrier translocase (also termed TIM22 complex) inserts proteins with multiple transmembrane segments into the inner membrane (3-9). The membrane potential across the inner membrane provides the driving force for both protein import pathways and is generated by the activity of the respiratory chain. Presequence-containing preproteins are directly transferred from the ...

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Section: Depletion Of Pc Impairs Protein Transport Into the Innermentioning

confidence: 92%

Phosphatidylcholine Affects Inner Membrane Protein Translocases of Mitochondria

Schuler¹,

Bartolomeo

Mårtensson

et al. 2016

Journal of Biological Chemistry

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“…The yeast strain expressing Por1 HA in the YPH499 background was generated by chromosomal integration of a coding region for a triple HA tag and a HIS3 cassette in front of the stop codon of POR1 by homologous recombination (22). Yeast cells were grown on yeast extract/peptone/glycerol medium (1% (w/v) yeast extract, 2% (w/v) bactopeptone, 3% (v/v) glycerol) at 24 -30°C.…”

Section: Methodsmentioning

confidence: 99%

“…After excessive washing with digitonin buffer containing 0.1% (w/v) digitonin, bound proteins were eluted under native conditions by incubation with 1 mg/ml HA peptides (Roche Diagnostics). Tom22 His -containing protein complexes were purified by nickel in complex with nitrilotriacetic acid-agarose (Qiagen, Germany) (22). Denatured proteins were analyzed by SDS-PAGE, and native samples were subjected to BN-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Cryo-slicing Blue Native-Mass Spectrometry (csBN-MS), a Novel Technology for High Resolution Complexome Profiling

Müller

Bildl

Haupt

et al. 2016

Molecular & Cellular Proteomics

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Blue native (BN)1 -PAGE and its colorless variant, colorless native PAGE, were originally developed by Schä gger and co-workers as end point separation methods for characterization of solubilized mitochondrial membrane protein (super-)-complexes under close-to-native conditions (1-3). Subsequently, native gel electrophoresis became the method of choice for first dimension separation followed by second dimension SDS-PAGE in two-dimensional gel-based proteomic analyses (2D-BN) of membrane protein complexes. After staining of the gel-separated proteins, protein spots are individually analyzed by different mass spectrometric methods, and the identified proteins were assigned to complexes based on their co-migration pattern (2D-BN-MS (4)). However, these 2D-BN-MS approaches exhibit the following severe shortcomings: (i) they are critically dependent on the staining properties of individual proteins; (ii) the size resolution of protein complexes is low; and (iii) the assignment of identified proteins to spots and complexes may be ambiguous. Therefore, application of 2D-BN-MS has remained largely restricted to the characterization of highly abundant and well defined membrane protein complexes such as complexes I-V of the respiratory chain in mitochondria (5-7), photosynthetic complexes (8 -10), or viruses (11).In a first attempt to overcome these shortcomings of 2D-BN-MS, Wessels et al. (12) coupled BN-PAGE separation more directly to MS analysis by manually cutting the gel lane into 24 slices/sections of about 2 mm width that were separately digested and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Their study on HEK cell mitochondria identified 59 of the 90 canonical subunits of the oxidative respiratory chain (OXPHOS) complexes I-V. The respective protein abundance profiles (based on standard label-free quantification) showed clustering of their peak maxima into the expected complexes I-V. Since then, this onedimensional BN-MS methodology has been gradually improved with respect to quality of the native gel separation, LC-MS/MS sensitivity, and robustness of the quantitative evaluation. Thus, two recent studies on human mitochondrial preparations (each analyzing two BN separations in 60 and 24 slices, respectively) reported identification and hierarchical profile clustering of 464 (13)

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“…Yeast Strains and Growth Conditions-The yeast wild-type strains YPH499, YPH499 arg4⌬, and JK9 -3d (the wild type for hsp10ts and hsp60ts); the wild-type strain for ssc1-42; and the mutant strains mtHsp70 His , ssc1-42, hsp10ts, and hsp60ts have been described before (39,44,54). For SILAC-based mass spectrometric analysis of mtHsp70 His purification, a kanMX4 cassette was integrated into the ARG4 locus by homologous recombination in the yeast strain expressing mtHsp70 His .…”

Section: Methodsmentioning

confidence: 99%

“…So far, it has been reported that mtHsp70 is required for the import of Hsp60, but a potential role of the chaperone in the formation of Hsp60 complexes has not been addressed (43). To distinguish between the role of mtHsp70 in import or folding and assembly of the Hsp60 precursors, we used a temperature-sensitive mutant of mtHsp70, ssc1-42 (54). The ssc1-42 mutant mtHsp70 harbors six point mutations that are distributed randomly in the ATPase, substrate-binding, and C-terminal domains of the chaperone (I101T, I114N, I485L, N560D, V588A, and K626R).…”

Section: Mthsp70 Promotes the Formation Of Hsp60mentioning

confidence: 99%