Electrophysiological Approaches to the Study of Protein Translocation in Mitochondria

Grigoriev, Sergey M.; Muro, Concepción; Dejean, Laurent M.; Campo, María Luisa; Martínez-Caballero, Sonia; Kinnally, Kathleen W.

doi:10.1016/s0074-7696(04)38005-8

Cited by 28 publications

(36 citation statements)

References 86 publications

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“…In addition, the channel of TIM23 was entirely independent of Tim22p. These results confirm our previous work in which the only channel activity related to protein import in the inner membrane of mitochondria was that of TIM23 (6,7,29).…”

Section: Decreasing Tim22p Does Not Affect the Electrical Behavior Ofsupporting

confidence: 92%

“…P2 was dissolved in 0.2% Me 2 SO. The presequence signal peptide (yCoxIV [1][2][3][4][5][6][7][8][9][10][11][12][13] ) was based on amino acids 1-13 (MLSLRQSIRFFKP) from the amino terminus of the cyto-chrome oxidase subunit IV of S. cerevisiae and was dissolved in H 2 O. Peptide composition and purity were assessed by high performance liquid chromatography and mass spectrometry and were typically Ͼ90% pure.…”

Section: Isolation Of Mitochondria and Preparation Of Mitoplasts Andmentioning

confidence: 99%

“…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][3][4][5][6][7].…”

mentioning

confidence: 99%

“…Section 1734 solely to indicate this fact. 1 genetic alterations of these two translocases has been essential to tackling their structure-function relationships and to pin down their subtle differences (6,7,19). Yet, the existence of a channel associated to the TIM22 translocase has been elusive and remains more arguable, as its function relates to the insertion of multispanning proteins into the inner membrane of mitochondria.…”

mentioning

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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Section: Decreasing Tim22p Does Not Affect the Electrical Behavior Ofsupporting

confidence: 92%

Section: Isolation Of Mitochondria and Preparation Of Mitoplasts Andmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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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“…The polymer exclusion method was used to measure the pore size of MAC, 23 as has been performed with other channels [31][32][33][34][35] MAC with a conductance of 4 nS is permeable to 10 and 17 kDa, but not 45 and 71 kDa dextran, as there is no current decrease upon introduction of the larger polymers (shown in Figure 1c). The polymer exclusion method indicates that MAC with conductances between 1.5 and 5 nS have pore sizes of 2.9-7.6 nm, which should be large enough to allow the passage of B3 nm cytochrome c. 36 Is MAC the Cytochrome c Release Channel?…”

Section: Electrophysiological Characterization Of Macmentioning

confidence: 99%

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

2006

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Apoptosis is a phenomenon fundamental to higher eukaryotes and essential to mechanisms controlling tissue homeostasis. Bcl-2 family proteins tightly control this cell death program by regulating the permeabilization of the mitochondrial outer membrane and, hence, the release of cytochrome c and other proapoptotic factors. Mitochondrial apoptosisinduced channel (MAC) is the mitochondrial apoptosisinduced channel and is responsible for cytochrome c release early in apoptosis. MAC activity is detected by patch clamping mitochondria at the time of cytochrome c release. The Bcl-2 family proteins regulate apoptosis by controlling the formation of MAC. Depending on cell type and apoptotic inducer, Bax and/or Bak are structural component(s) of MAC. Overexpression of the antiapoptotic protein Bcl-2 eliminates MAC activity. The focus of this review is a biophysical characterization of MAC activity and its regulation by Bcl-2 family proteins, and ends with some discussion of therapeutic targets.

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Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins

2006

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Apoptosis is a phenomenon fundamental to higher eukaryotes that is integral to such diverse cellular processes as tissue homeostasis, organogenesis, and response to toxins. The release from mitochondria of apoptotic factors such as cytochrome c is a key step during apoptosis of most cells. Cytochrome c release occurs through the MAC (mitochondrial apoptosis-induced channel), a pore which forms in the mitochondrial outer membrane during early apoptosis and is exquisitely regulated by the Bcl-2 family of proteins. This unit presents basic and advanced tools for detecting MAC and defining its regulation by Bcl-2 family proteins and pharmacological agents. Protocols include the use of time-lapse video-microscopy to monitor the onset of apoptosis in living cells and patch-clamp techniques for mitochondria or proteoliposomes containing mitochondrial proteins, which allow direct detection of MAC. These approaches enable an evaluation of the role of MAC and mitochondria in apoptosis of a variety of cell types by many inducers.

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Electrophysiological Approaches to the Study of Protein Translocation in Mitochondria

Cited by 28 publications

References 86 publications

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

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

Is MAC the knife that cuts cytochrome c from mitochondria during apoptosis?

Detection of the Mitochondrial Apoptosis‐Induced Channel (MAC) and Its Regulation by Bcl‐2 Family Proteins

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