A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments

Previously we showed that xenobiotic-inducible cytochrome P450 (CYP) proteins are bimodally targeted to the endoplasmic reticulum and mitochondria. In the present study, we investigated the mechanism of delivery of chimeric signal-containing CYP proteins to the peripheral and channel-forming mitochondrial outer membrane translocases (TOMs). CYP؉33/1A1 and CYP2B1 did not require peripheral TOM70, TOM20, or TOM22 for translocation through the channel-forming TOM40 protein. In contrast, CYP؉5/1A1 and CYP2E1 were able to bypass TOM20 and TOM22 but required TOM70. CYP27, which contains a canonical cleavable mitochondrial signal, required all of the peripheral TOMs for its mitochondrial translocation. We investigated the underlying mechanisms of bypass of peripheral TOMs by CYPs with chimeric signals. The results suggested that interaction of CYPs with Hsp70, a cytosolic chaperone involved in the mitochondrial import, alone was sufficient for the recognition of chimeric signals by peripheral TOMs. However, sequential interaction of chimeric signal-containing CYPs with Hsp70 and Hsp90 resulted in the bypass of peripheral TOMs, whereas CYP27 interacted only with Hsp70 and was not able to bypass peripheral TOMs. Our results also show that delivery of chimeric signal-containing client proteins by Hsp90 required the cytosol-exposed N-terminal 143 amino acids of TOM40. TOM40 devoid of this domain was unable to bind CYP proteins. These results suggest that, compared with the unimodal mitochondria-targeting signals, the chimeric mitochondriatargeting signals are highly evolved and dynamic in nature.Protein targeting to different subcellular compartments is directed by specific N-terminal or internal signals, which serve as destination-specific mail delivery codes (1, 2). The signal sequences of proteins targeted to different membrane compartments, such as the endoplasmic reticulum (ER), 2 peroxisomes, and mitochondria, vary markedly in terms of amino acid sequence, hydrophobicity, and secondary structure and interact with distinctly different sets of carrier proteins, receptors, and protein translocator complexes (3-8). These observations have led to a widely accepted view that most protein targeting signals are unimodal in nature, which in turn restricts the destination of a given protein to a single subcellular compartment.ER-targeted proteins contain a distinct N-terminal hydrophobic signal for binding to a signal recognition particle, which in turn targets the emerging nascent chains to the ER (9). With certain exceptions (1, 10), ER targeting is thought to be cotranslational. Current models of protein targeting imply that the ER or mitochondrial destination of a protein is determined at the pretranslational level by virtue of the signal sequence that the protein carries. Many mitochondria-and ER-targeted proteins are encoded by a distinct set of genes. In a limited number of cases, characteristic mitochondrial targeting sequences are generated by differential expression of the gene using either alternate transcription/tr...

Section: Targeting Of Cyp Proteins To Mitochondria In Intact Cellsmentioning

confidence: 51%

An Unusual TOM20/TOM22 Bypass Mechanism for the Mitochondrial Targeting of Cytochrome P450 Proteins Containing N-terminal Chimeric Signals

Anandatheerthavarada

Sepuri²,

Biswas³

et al. 2008

“…For example, proteins containing a presequence are imported in a Tom20-dependent manner, whereas carrier proteins and proteins containing multiple ␣-helical transmembrane segments are imported in a Tom70-dependent manner. Moreover, ␤-barrel proteins are integrated into the mitochondrial outer membrane by the cooperation of Tom20-and Tom70-dependent processes (32,57). Using immunoprecipitation in the presence of a crosslinker and with Tom20 and Tom22 under native conditions, Lazarou et al reported that PINK1 interacts with Tom20, Tom22, Tom40, and Tom70 (18).…”

Section: Discussionmentioning

confidence: 99%

A Dimeric PINK1-containing Complex on Depolarized Mitochondria Stimulates Parkin Recruitment

Okatsu

Uno

Koyano

et al. 2013

Self Cite

196

166

Background: PINK1 functions on depolarized mitochondria. However, details regarding its mechanism remain limited. Results: We reveal the formation of a high molecular weight complex composed of two phosphorylated PINK1 molecules that stimulates Parkin recruitment. Conclusion:The dimeric PINK1-containing complex is important for mitochondrial quality control. Significance: The PINK1 molecular process is reminiscent of receptor kinase dimerization in signal transduction.

“…However, the identified MTS of APE1 in the present study is located in the C terminus, which is more similar to tailanchored preproteins. The mitochondrial tail-anchored preproteins, including Bcl-2 and Tom20, were inserted into the mitochondrial outer membranes via a C-terminal transmembrane domain (28,29). Although the exact localization of APE1 in the mitochondria has not been well characterized, according to its function in mitochondrial DNA repair, its distribution should accompany the mitochondrial DNA in the mitochondrial matrix.…”

Section: Discussionmentioning

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.