Accessing mitochondrial protein import in living cells by protein microinjection
Accessing mitochondrial protein import in living cells by protein microinjection
Abstract 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.
Borshchevskiy Valentin、Bogorodskiy Andrey、Maliar Nina、Schulga Alexey、Altschmied Joachim、Katranidis Alexandros、Mishin Alexey、Gensch Thomas、B¨1ldt Georg、Jakobs Philipp、Maslov Ivan、Okhrimenko Ivan、von Ameln Florian、Gordeliy Valentin、Haendeler Judith、Burkatovskiy Dmitry、Voos Wolfgang
Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology||Institute of Biological Information Processing (IBI-7: Structural Biochemistry)||JuStruct: J¨1lich Center for Structural BiologyResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyMolecular Immunology Laboratory, Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of SciencesIUF-Leibniz Research Institute for Environmental MedicineInstitute of Biological Information Processing (IBI-6: Cellular Structural Biology)Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyInstitute of Biological Information Processing (IBI-1: Molecular and Cellular Physiology)Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyCentral Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, University of D¨1sseldorfResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyIUF-Leibniz Research Institute for Environmental MedicineResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology||Institute of Biological Information Processing (IBI-7: Structural Biochemistry)||JuStruct: J¨1lich Center for Structural Biology||Institut de Biologie Structurale (IBS), Universit¨| Grenoble AlpesCentral Institute of Clinical Chemistry and Laboratory Medicine, Medical Faculty, University of D¨1sseldorfResearch Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and TechnologyInstitute for Biochemistry and Molecular Biology (IBMB), University of Bonn
细胞生物学生物化学分子生物学
Borshchevskiy Valentin,Bogorodskiy Andrey,Maliar Nina,Schulga Alexey,Altschmied Joachim,Katranidis Alexandros,Mishin Alexey,Gensch Thomas,B¨1ldt Georg,Jakobs Philipp,Maslov Ivan,Okhrimenko Ivan,von Ameln Florian,Gordeliy Valentin,Haendeler Judith,Burkatovskiy Dmitry,Voos Wolfgang.Accessing mitochondrial protein import in living cells by protein microinjection[EB/OL].(2025-03-28)[2025-05-15].https://www.biorxiv.org/content/10.1101/2020.09.30.317412.点此复制
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