Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.

Rojo, Elena E.; Stuart, Rosemary A.; Neupert, Walter

doi:10.1002/j.1460-2075.1995.tb07350.x

Cited by 70 publications

(78 citation statements)

References 43 publications

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“…Saturation of mouse liver mitochondria TOM and TIM23 complexes with cold SU9-DHFR (dihydrofolate reductase) and B2Δ19-DHFR inhibited in a dose-dependent manner the import of 35 S-Met-labeled SU9-DHFR 23,[32][33][34][35] used throughout this work as a positive control (Supplementary Figure 2A). This inhibition was even more pronounced when cold SU9-DHFR and B2Δ19-DHFR were stabilized with methotrexate (MTX) 23 (Supplementary Figure 2B).…”

Section: Resultsmentioning

confidence: 97%

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

et al. 2017

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We have found that granzyme B (GB)-induced apoptosis also requires reactive oxygen species resulting from the alteration of mitochondrial complex I. How GB, which does not possess a mitochondrial targeting sequence, enter this organelle is unknown. We show that GB enters the mitochondria independently of the translocase of the outer mitochondrial membrane complex, but requires instead Sam50, the central subunit of the sorting and assembly machinery that integrates outer membrane β-barrel proteins. Moreover, GB breaches the inner membrane through Tim22, the metabolite carrier translocase pore, in a mitochondrial heat-shock protein 70 (mtHsp70)-dependent manner. Granzyme A (GA) and caspase-3 use a similar route to the mitochondria. Finally, preventing GB from entering the mitochondria either by mutating lysine 243 and arginine 244 or depleting Sam50 renders cells more resistant to GB-mediated reactive oxygen species and cell death. Similarly, Sam50 depletion protects cells from GA-, GM-and caspase-3-mediated cell death. Therefore, cytotoxic molecules enter the mitochondria to induce efficiently cell death through a noncanonical Sam50-, Tim22-and mtHsp70-dependent import pathway.Cytotoxic lymphocytes eradicate pathogen-infected or transformed cells mainly through the cytotoxic granule pathway. [1][2][3][4] Among the five human granzymes (A, B, H, K and M), granzyme B (GB) triggers cell death in both a caspasedependent and caspase-independent manner, although human and mouse GB differ in their requirement for caspase activation. 5-10 GB-induced cell death also involves Bid/Bax/ Bak-mediated mitochondrial outer membrane permeabilization for the release of the apoptogenic factors cytochrome c, Smac/Diablo, Endo G, HtrA2 and AIF. 1,9-17 We have recently reported that mitochondrial reactive oxygen species (ROS) have a critical role in GB pathway by amplifying apoptogenic factor release, oligonucleosomal DNA fragmentation and lysosomal membrane rupture. 18 Mitochondrial ROS resulted from GB-mediated cleavage of NDUFV1, NDUFS1 and NDUFS2, three subunits of mitochondrial complex I. 18 How GB that does not possess a mitochondrial targeting sequence enters the mitochondria is not known. The mitochondrial nucleoid encodes only for 13 structural proteins, while the rest of the mitochondrial proteome is nuclear-encoded and transported to the mitochondria through a sophisticated protein import machinery. [19][20][21][22][23][24][25][26][27][28] The translocase of the outer mitochondrial membrane (TOM), the entry gate to the mitochondria, 20 passes on the cargos to the sorting and assembly machinery (SAM) complex, to the mitochondrial intermembrane space assembly (MIA) complex and to the translocases of the inner mitochondrial membrane TIM22 or TIM23 complexes for delivery to the outer membrane, the intermembrane space, the inner membrane or the matrix, respectively. [19][20][21][22]25,26,28 Unexpectedly, we found that GB mitochondrial entry is independent of the TOM-TIM23 complexes, but requires instead the Sam50 and Tim22 chann...

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Section: Resultsmentioning

confidence: 97%

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

et al. 2017

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“…Import was performed as described previously (2,20). Import in the presence EDTA and o-phenanthroline and their subsequent chase by the addition of Mg 2ϩ /Mn 2ϩ was performed as described previously (2).…”

Section: Isolation Of Mitochondria and Protein Importmentioning

confidence: 99%

“…Following maturation by the mitochondrial-processing peptidase (MPP), 1 insertion into the inner membrane leading to export of hydrophilic domains to the intermembrane space occurs. Export from the matrix requires a membrane potential (1)(2)(3) and, at least for those proteins that contain their N termini in the intermembrane space, is mediated by the Oxa1p export machinery (4 -6).…”

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confidence: 99%

“…To study the importance of the charge distribution in the process of mitochondrial N-terminal tail export, we analyzed the sorting of a fusion protein, preSu9(1-112)-DHFR (2,15). This protein consists of the N-terminal region of subunit 9 from the F 1 F O -ATPase (Su9) of Neurospora crassa encompassing the presequence and the first half of the mature part of the protein containing a single transmembrane segment.…”

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confidence: 99%

“…As previously shown, preSu9(1-112)-DHFR is initially imported into the mitochondrial matrix, where it is processed by MPP. The N-tail becomes exported across the inner membrane in a membrane potential and Oxa1p-dependent fashion to give rise to an N out -C in topology (2,6). The exported N-terminal tail of mSu9(1-112)-DHFR bears a net negative charge.…”

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N-terminal Tail Export from the Mitochondrial Matrix

Rojo

Guiard²,

Neupert

et al. 1999

Journal of Biological Chemistry

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Export of N-terminal tails of mitochondrial inner membrane proteins from the mitochondrial matrix is a membrane potential-dependent process, mediated by the Oxa1p translocation machinery. The hydrophilic segments of these membrane proteins, which undergo export, display a characteristic charge profile where intermembrane space-localized segments bear a net negative charge, whereas those remaining in the matrix have a net positive one. Using a model protein, preSu9(1-112)-dihydrofolate reductase (DHFR), which undergoes Oxa1p-mediated N-tail export, we demonstrate here that the net charge of N-and C-flanking regions of the transmembrane domain play a critical role in determining the orientation of the insertion process. The N-tail must bear a net negative charge to be exported to the intermembrane space. Furthermore, a net positive charge of the C-terminal region supports this N-tail export event. These data provide experimental evidence that protein export in mitochondria adheres to the "positive-inside" rule, described for secindependent sorting of membrane proteins in prokaryotes. We propose here that the importance of a charge profile reflects a need for specific protein-protein interactions to occur in the export reaction, presumably at the level of the Oxa1p export machinery.

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Protein Targeting, Intracellular

Hartmann¹

2002

Encyclopedia of Molecular Biology

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Conservative sorting of F0-ATPase subunit 9: export from matrix requires delta pH across inner membrane and matrix ATP.

Cited by 70 publications

References 43 publications

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

Granzyme B enters the mitochondria in a Sam50-, Tim22- and mtHsp70-dependent manner to induce apoptosis

N-terminal Tail Export from the Mitochondrial Matrix

Protein Targeting, Intracellular

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