Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.
GFP
SNAP-tag
fluorescence microscopy
microinjection
mitochondria
mitochondrial protein import
Journal
Frontiers in cell and developmental biology
ISSN: 2296-634X
Titre abrégé: Front Cell Dev Biol
Pays: Switzerland
ID NLM: 101630250
Informations de publication
Date de publication:
2021
2021
Historique:
received:
21
04
2021
accepted:
15
06
2021
entrez:
26
7
2021
pubmed:
27
7
2021
medline:
27
7
2021
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 34307376
doi: 10.3389/fcell.2021.698658
pmc: PMC8292824
doi:
Types de publication
Journal Article
Langues
eng
Pagination
698658Informations de copyright
Copyright © 2021 Bogorodskiy, Okhrimenko, Maslov, Maliar, Burkatovskii, von Ameln, Schulga, Jakobs, Altschmied, Haendeler, Katranidis, Sorokin, Mishin, Gordeliy, Büldt, Voos, Gensch and Borshchevskiy.
Déclaration de conflit d'intérêts
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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