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Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

ABSTRACT: The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP and Tim23 which are inserted into the inner membrane and, at the same time, are spanning either the TOM complex or are integrated into the outer membrane. Our results demonstrate that the TOM complex, depending on sequence determinants in the precursors, can act both as a protein conducting pore and as an insertase mediating lateral release into the outer membrane.

SUBMITTER: Harner M

PROVIDER: S-EPMC3160657 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

Harner Max M Neupert Walter W Deponte Marcel M

The EMBO journal 20110715 16

The TOM complex of the outer membrane of mitochondria is the entry gate for the vast majority of precursor proteins that are imported into the mitochondria. It is made up by receptors and a protein conducting channel. Although precursor proteins of all subcompartments of mitochondria use the TOM complex, it is not known whether its channel can only mediate passage across the outer membrane or also lateral release into the outer membrane. To study this, we have generated fusion proteins of GFP an ...[more]

PMID: 21765393

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Project description:Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded β-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded β-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.

Project description:Mitochondria originated from α-proteobacterial endosymbionts, and the endosymbiont-to-organelle transition is tightly linked to the establishment of protein import pathways. The initial import of most proteins is mediated by the translocase of the outer membrane (TOM). Although TOM is common to all forms of mitochondria, recent studies have shown diversity of TOM subunits between main eukaryotic lineages. However, experimental knowledge is currently limited to a few model organisms. Here, we analysed the TvTOM complex in hydrogenosomes, an anaerobic form of mitochondria found in the excavate Trichomonas vaginalis. Using the pore-forming TvTom40-2 as bait, we immunoprecipitated the high molecular weight TvTOM complex both under crosslinking and native conditions and performed label-free quantitative mass spectrometry (LFQ-MS) to identify the components of the TvTOM complex. Identification of the components of the hydrogenosomal TOM complex To identify interaction partners for TvTom40-2 in the TOM complex, we performed co-immunoprecipitations (coIPs) of HA-tagged TvTom40-2 under protein crosslinking and under native conditions, and the eluted proteins were analyzed using label-free quantitative (LFQ) mass spectrometry. CoIPs using anti-HA antibody (Ab) were performed with hydrogenosomes isolated from both the strain expressing HA-tagged TvTom40-2 and the wild type strain, as a negative control. The analysis revealed 50 and 36 proteins that were enriched with HA-tagged TvTom40-2 under crosslinking and native conditions respectively. As TOM proteins are embedded in the hydrogenosomal outer membrane, we searched for proteins with TMDs in the datasets using TMHMM and found 19 and 13 proteins for crosslinking and native coIPs respectively. The intersection between the two datasets and the hydrogenosomal membrane proteome (Rada et al 2011) contained five TvTom40 isoforms, two TA proteins named Homp36 and Homp19 (Homp, Hydrogenosomal outer membrane protein), Sam50 and its paralogue Sam50p, and Hmp35. In addition, the intersection between the coIP dataset under crosslinking conditions and the membrane proteome contained two more TA proteins, Homp38 and Homp46. Based on these results, we selected Homp36 which was previously shown to reside in the hydrogenosomal outer membrane (Rada et al 2011) for the reciprocal coIPs. Proteins enriched in the HA-tagged Homp36 coIPs under crosslinking conditions included multiple isoforms of TvTom40, Sam50, Hmp35, Homp38, Homp46 and Homp19, and multiple isoforms of TvTom40, Sam50 and Hmp35 were enriched under native conditions. Altogether, the coIP and MS data indicated four TA candidate proteins, Homp19, Homp36, Homp38 and Homp46, and two β-barrel proteins, Sam50 and Hmp35. InterProScan predicted that Homp36, Homp38 and Homp46 carry an N-terminal Hsp20-like chaperone domain, three TPR-like domains and a C-terminal transmembrane helix. This domain architecture resembles the recently reported ATOM69 in T. brucei (Mani et al 2015). Indeed, HHpred searches using Homp36 and Homp38 as queries against the T. brucei proteome revealed ATOM69 as the first hit, with E values of 4.9e-17 and 2.3e-11 respectively. HHpred searches with Homp46 recognized various proteins with TPR domains, whereas no significant homology was observed for Homp19.

Dataset Information

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

Publications

Lateral release of proteins from the TOM complex into the outer membrane of mitochondria.

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