Preprotein Translocase of the Outer Mitochondrial Membrane: Reconstituted Tom40 Forms a Characteristic TOM Pore

Background: Nearly all nascent mitochondrial proteins are transported by the translocase of the outer membrane (TOM) complex. Results:The core Tom40 ␤-barrel domain exhibits four conductive levels and three distinct substrate binding affinities. Conclusion: Tom40 interactions with presequence substrates depend upon the channel's conformation. Significance: Conformational rearrangements in Tom40 may regulate substrate interactions.

Section: Discussionmentioning

confidence: 63%

Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40

Kuszak

Jacobs

Gurnev

et al. 2015

“…Indeed, the channels associated with TOM and TIM23 have been identified (18,(33)(34)(35). Two of the main lines of evidence connecting a particular mitochondrial channel with any of these translocases rely on the effect of amino-terminal signal sequences and the outcome of different mutations involving the core components of both complexes (18,19,29,30).…”

Section: Discussionmentioning

confidence: 99%

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

Peixoto

Graña

Roy

et al. 2007

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...

“…Like the proteins destined for other subcellular organelles, the mitochondrially targeted proteins possess targeting signals within their primary or secondary structure that direct them to the organelle with the assistance of elaborate protein translocating and folding machines (12,13). The nucleus-encoded mitochondrially targeted preproteins are recognized by the receptors on the mitochondrial surface and are subsequently translocated across the outer mitochondrial membranes through a general import pore that assembles into a high molecular weight complex, termed the preprotein translocase of the outer mitochondrial membrane (Tom) (13,14). The three Tom subunits, Tom20, Tom22, and Tom70, expose major portions on the cytosolic side of the outer membrane and function as import receptors for distinct classes of the preproteins in vivo and in organello (15).…”

mentioning

confidence: 99%

Identification and Characterization of Mitochondrial Targeting Sequence of Human Apurinic/Apyrimidinic Endonuclease 1

Zhong

Zhu

et al. 2010

Dually targeted mitochondrial proteins usually possess an unconventional mitochondrial targeting sequence (MTS), which makes them difficult to predict by current bioinformatics approaches. Human apurinic/apyrimidinic endonuclease (APE1) plays a central role in the cellular response to oxidative stress. It is a dually targeted protein preferentially residing in the nucleus with conditional distribution in the mitochondria. However, the mitochondrial translocation mechanism of APE1 is not well characterized because it harbors an unconventional MTS that is difficult to predict by bioinformatics analysis. Two experimental approaches were combined in this study to identify the MTS of APE1. First, the interactions between the peptides from APE1 and the three purified translocase receptors of the outer mitochondrial membrane (Tom) were evaluated using a peptide array screen. Consequently, the intracellular distribution of green fluorescent protein-tagged, truncated, or mutated APE1 proteins was traced by tag detection. The results demonstrated that the only MTS of APE1 is harbored within residues 289 -318 in the C terminus, which is normally masked by the intact N-terminal structure. As a dually targeted mitochondrial protein, APE1 possesses a special distribution pattern of different subcellular targeting signals, the identification of which sheds light on future prediction of MTSs.Human APE1 (apurinic/apyrimidinic endonuclease) is an important multifunctional protein that plays a central role in the cellular response to oxidative stress. The two major activities of APE1 are DNA repair and redox regulation of transcriptional factors. On one hand, APE1 functions as a critical ratelimiting enzyme in DNA base excision repair and accounts for nearly all of the AP site incision activities in cell extracts (1). On the other hand, APE1 also exerts unique redox activity to regulate the DNA binding affinity of certain transcriptional factors by controlling the redox status of their DNA-binding domain (2). Inhibition of the redox function of APE1 blocks murine endothelial cell growth and angiogenesis and also blocks the growth of human tumor cell lines (3). The biological importance of APE1 is highlighted by the finding that APE1 knockout mice exhibit an embryonic lethal phenotype (4). Although APE1 has long been labeled as a nuclear protein, a growing body of evidence has shown that the subcellular distribution of APE1 can be cytoplasmic in some cell types with high metabolic or proliferative rates, with predominant localization in the mitochondria and the endoplasmic reticulum (5-7). Recent mitochondrial proteomic studies have further confirmed the existence of APE1 in the mitochondria (8). Considering the importance of the mitochondria in cellular response to oxidative stress, the roles of APE1 in the mitochondria have been extensively investigated.