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  • 标题:A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface
  • 作者:Dominik P. Buser ; Kai D. Schleicher ; Cristina Prescianotto-Baschong
  • 期刊名称:Proceedings of the National Academy of Sciences
  • 印刷版ISSN:0027-8424
  • 电子版ISSN:1091-6490
  • 出版年度:2018
  • 卷号:115
  • 期号:27
  • 页码:E6227-E6236
  • DOI:10.1073/pnas.1801865115
  • 语种:English
  • 出版社:The National Academy of Sciences of the United States of America
  • 摘要:Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans -Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters’ homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans -Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.
  • 关键词:nanobodies ; retrograde transport ; tyrosine sulfation ; AP-1 ; mannose-6-phosphate receptors
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