The PINK1 kinase-driven ubiquitin ligase Parkin promotes mitochondrial protein import through the presequence pathway in living cells

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected pre-protein. This suggests that a substantial amount of mitochondrial translocase complexes was involved in co-translational protein import of endogenously expressed pre-proteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

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Accessing mitochondrial protein import in living cells by protein microinjection

10.1101/2020.09.30.317412 ◽

2020 ◽

Author(s):

Andrey Bogorodskiy ◽

Ivan Okhrimenko ◽

Ivan Maslov ◽

Nina Maliar ◽

Dmitry Burkatovskiy ◽

...

Keyword(s):

Real Time ◽

Protein Import ◽

Expression System ◽

Mitochondrial Protein ◽

Living Cells ◽

Mitochondrial Morphology ◽

Mitochondrial Protein Import ◽

Intact Cells ◽

Endogenous Expression ◽

Cellular Environment

AbstractMitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment. We developed a gentle microinjection procedure for fluorescent reporter proteins allowing a direct non-invasive study of protein transport in living cells. As a proof of principle, we visualized potential-dependent protein import into mitochondria inside intact cells in real-time. We validated that our approach does not distort mitochondrial morphology and preserves the endogenous expression system as well as mitochondrial protein translocation machinery. We observed that a release of nascent polypeptides chains from actively translating cellular ribosomes by puromycin strongly increased the import rate of the microinjected preprotein. This suggests that a substantial amount of mitochondrial translocase complexes were involved in co-translational protein import of endogenously expressed preproteins. Our protein microinjection method opens new possibilities to study the role of mitochondrial protein import in cell models of various pathological conditions as well as aging processes.

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Mistargeting of hydrophobic mitochondrial proteins activates a nucleus-mediated posttranscriptional quality control pathway in trypanosomes

10.1101/2021.08.13.456187 ◽

2021 ◽

Author(s):

Caroline E. Dewar ◽

Silke Oeljeklaus ◽

Jan Mani ◽

Wignand W. D. Mühlhäuser ◽

Bettina Warscheid ◽

...

Keyword(s):

Quality Control ◽

Ubiquitin Ligase ◽

Protein Import ◽

Nuclear Protein ◽

Mitochondrial Protein ◽

Protein A ◽

Mitochondrial Proteins ◽

Mitochondrial Protein Import ◽

Mitochondrial Quality Control ◽

Import Receptor

Mitochondrial protein import in the parasitic protozoan Trypanosoma brucei is mediated by the atypical outer membrane translocase, ATOM. It consists of seven subunits including ATOM69, the import receptor for hydrophobic proteins. Ablation of ATOM69, but not of any other subunit, triggers a unique quality control pathway resulting in the proteasomal degradation of non-imported mitochondrial proteins. The process requires a protein of unknown function, an E3 ubiquitin ligase and the ubiquitin-like protein (TbUbL1), which all are recruited to the mitochondrion upon ATOM69 depletion. TbUbL1 is a nuclear protein, a fraction of which is released to the cytosol upon triggering of the pathway. Nuclear release is essential as cytosolic TbUbL1 can bind mislocalised mitochondrial proteins and likely transfers them to the proteasome. Mitochondrial quality control has previously been studied in yeast and metazoans. Finding such a pathway in the highly diverged trypanosomes suggests such pathways are an obligate feature of all mitochondria.

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