TY - JOUR T1 - Accessing mitochondrial protein import in living cells by protein microinjection JF - bioRxiv DO - 10.1101/2020.09.30.317412 SP - 2020.09.30.317412 AU - Andrey Bogorodskiy AU - Ivan Okhrimenko AU - Ivan Maslov AU - Nina Maliar AU - Dmitry Burkatovskiy AU - Florian von Ameln AU - Alexey Schulga AU - Philipp Jakobs AU - Joachim Altschmied AU - Judith Haendeler AU - Alexandros Katranidis AU - Alexey Mishin AU - Valentin Gordeliy AU - Georg Büldt AU - Wolfgang Voos AU - Thomas Gensch AU - Valentin Borshchevskiy Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/10/01/2020.09.30.317412.abstract N2 - 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 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.Competing Interest StatementThe authors have declared no competing interest. ER -