Comparison of the TIM and TOM Channel Activities of the Mitochondrial Protein Import Complexes

Muro, Concepción; Grigoriev, Serguei M.; Pietkiewicz, Dawn; Kinnally, Kathleen W.; Campo, María Luisa

doi:10.1016/s0006-3495(03)70024-1

Cited by 54 publications

(52 citation statements)

References 47 publications

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“…Mitochondria were isolated from Saccharomyces cerevisiae grown in lactose media and harvested in log phase as described (35)(36)(37). Wild type (strain M3) and a strain of yeast, M22-2 (por1) in which por1 (YVDAC1) had been deleted (38), were used as indicated.…”

Section: Methodsmentioning

confidence: 99%

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Jonas

Hickman

Chachar

et al. 2004

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

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Neuronal death is often preceded by functional alterations at nerve terminals. Anti-and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. Ntruncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltagedependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway.

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Section: Methodsmentioning

confidence: 99%

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Jonas

Hickman

Chachar

et al. 2004

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

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“…The two channel activities of these translocases are remarkably similar and highly conserved among mammals and yeast, in accordance with their analogous function in protein translocation (6,18). Slightly cation-selective and highly conductive channels of ϳ1000 pS with a predominant half-open state of ϳ500 pS have been described in proteoliposomes containing purified outer or inner membranes.…”

mentioning

confidence: 87%

“…Mitochondria were isolated from logarithmically growing cells as previously described (18). Homogenization buffer was 0.6 M sorbitol, 10 mM Tris, pH 7.4, 1 mM EDTA, 0.2% bovine serum albumin, 1 mM phenylmethylsulfonyl fluoride containing protease inhibitor mixture (P8215; Sigma).…”

Section: Isolation Of Mitochondria and Preparation Of Mitoplasts Andmentioning

confidence: 99%

“…Membrane purity was routinely assessed, and cross-contamination with the outer membrane was typically Ͻ10%. Inner membranes were reconstituted into giant proteoliposomes by dehydration-rehydration as previously described (18,25) using soybean L-␣-phosphatidylcholine (Sigma Type IV-S).…”

Section: Isolation Of Mitochondria and Preparation Of Mitoplasts Andmentioning

confidence: 99%

“…Filtration was 2 kHz with 5 kHz sampling for all analysis and current traces shown, unless otherwise stated. Permeability ratios were calculated from the reversal potential in the presence of a 150:30 mM KCl gradient as previously described (18). Signal peptides were introduced in the pipette.…”

Section: Isolation Of Mitochondria and Preparation Of Mitoplasts Andmentioning

confidence: 99%

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Awaking TIM22, a Dynamic Ligand-gated Channel for Protein Insertion in the Mitochondrial Inner Membrane

Peixoto

Graña

Roy

et al. 2007

Journal of Biological Chemistry

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Aqueous channels are at the core of the translocase of the outer membrane (TOM) and the translocase of the inner membrane for the transport of preproteins (TIM23), the translocases mediating the transport of proteins across the outer and inner mitochondrial membranes. Yet, the existence of a channel associated to the translocase of the inner membrane for the insertion of multitopic protein (TIM22) complex has been arguable, as its function relates to the insertion of multispanning proteins into the inner membrane. For the first time, we report conditions for detecting a channel activity associated to the TIM22 translocase in organelle, i.e. intact mitoplasts. An internal signal peptide in the intermembrane space of mitochondria is a requisite to inducing this channel, which is otherwise silent. The channel showed slightly cationic and high conductance activity of 1000 pS with a predominant half-open substate. Despite their different composition, the channels of the three mitochondrial translocases were thus remarkably similar, in agreement with their common task as pores transiently trapping proteins en route to their final destination. The opening of the TIM22 channel was a step-up process depending on the signal peptide concentration. Interestingly, low membrane potentials kept the channel fully open, providing a threshold level of the peptide is present. Our results portray TIM22 as a dynamic channel solely active in the presence of its cargo proteins. In its fully open conformation, favored by the combined action of internal signal peptide and low membrane potential, the channel could embrace the in-transit protein. As insertion progressed and initial interaction with the signal peptide faded, the channel would close, sustaining its role as a shunt that places trapped proteins into the membrane.Most cells depend upon mitochondria to accomplish their programmed role. Along with mitochondria's own biogenesis, functioning and death entirely rely on protein translocation. The elevated protein content of mitochondria (ϳ700 different proteins in yeast (1)) and the four different compartments enclosed by their two membranes add to the complexity of protein trafficking pathways in this organelle. In addition, the need to maintain a low permeability of the inner membrane for coupling oxidative phosphorylation must be balanced with the passage of large molecules as proteins through water-filled channels. Focused on the route to their final destination inside mitochondria, three multisubunit complexes or translocases are depicted as the main machineries for specific recognition, import, and sorting of precursor proteins synthesized in the cytoplasm (for review, see Refs. 2-7).The translocase of the outer membrane (TOM) 3 is the common gate for the transport of every mitochondrial protein across the outer membrane (for review, see Ref. 8), and the TIM23 translocase imports matrix-targeted preproteins with cleavable amino-terminal presequences. Few proteins of the inner membrane (IM) containing a single transmembrane seg...

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Unique Mitochondrial Ion Channels: Roles in Synaptic Transmission and Programmed Cell Death

Jonas¹

2008

Structure, Function, and Modulation of Neuronal Voltagegated Ion Channels

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Comparison of the TIM and TOM Channel Activities of the Mitochondrial Protein Import Complexes

Cited by 54 publications

References 47 publications

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Awaking TIM22, a Dynamic Ligand-gated Channel for Protein Insertion in the Mitochondrial Inner Membrane

Unique Mitochondrial Ion Channels: Roles in Synaptic Transmission and Programmed Cell Death

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