A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing
| العنوان: | A clathrin coat assembly role for the muniscin protein central linker revealed by TALEN-mediated gene editing |
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| المؤلفون: | Li Ma, Anupma Jha, Linton M. Traub, Balraj Doray, Perunthottathu K. Umasankar, Simon C. Watkins, James R. Thieman |
| المصدر: | eLife eLife, Vol 3 (2014) |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | Phosphatidylinositol 4,5-Diphosphate, Endocytic cycle, Biochemistry, Mice, TALEN, Biology (General), Conserved Sequence, Phylogeny, 0303 health sciences, adaptor, General Neuroscience, 030302 biochemistry & molecular biology, General Medicine, Cell biology, Medicine, Clathrin adaptor proteins, Research Article, QH301-705.5, Science, Recombinant Fusion Proteins, Molecular Sequence Data, Adaptor Protein Complex 2, Biology, Endocytosis, Fatty Acid-Binding Proteins, Clathrin, General Biochemistry, Genetics and Molecular Biology, Exocytosis, 03 medical and health sciences, Allosteric Regulation, clathrin, Animals, Humans, endocytosis, human, 030304 developmental biology, General Immunology and Microbiology, Clathrin coat assembly, Base Sequence, Cell Membrane, E. coli, Membrane Proteins, Receptor-mediated endocytosis, Cell Biology, Endonucleases, Protein Structure, Tertiary, Rats, Membrane protein, Genetic Loci, biology.protein, Trans-Activators, RNA Editing, Peptides, HeLa Cells |
| الوصف: | Clathrin-mediated endocytosis is an evolutionarily ancient membrane transport system regulating cellular receptivity and responsiveness. Plasmalemma clathrin-coated structures range from unitary domed assemblies to expansive planar constructions with internal or flanking invaginated buds. Precisely how these morphologically-distinct coats are formed, and whether all are functionally equivalent for selective cargo internalization is still disputed. We have disrupted the genes encoding a set of early arriving clathrin-coat constituents, FCHO1 and FCHO2, in HeLa cells. Endocytic coats do not disappear in this genetic background; rather clustered planar lattices predominate and endocytosis slows, but does not cease. The central linker of FCHO proteins acts as an allosteric regulator of the prime endocytic adaptor, AP-2. By loading AP-2 onto the plasma membrane, FCHO proteins provide a parallel pathway for AP-2 activation and clathrin-coat fabrication. Further, the steady-state morphology of clathrin-coated structures appears to be a manifestation of the availability of the muniscin linker during lattice polymerization. DOI: http://dx.doi.org/10.7554/eLife.04137.001 eLife digest Cells can take proteins and other molecules that are either embedded in, or attached to, their surface membrane and move them inside via a process called endocytosis. This process often involves a protein called clathrin working together with numerous other proteins. Early on, a complex of four proteins, called the adaptor protein-2 complex, interacts with both the ‘cargo’ molecules that are to be taken into the cell, and the cell membrane. Clathrin molecules then assemble into an ordered lattice-like coat, on top of the adaptor protein complex layer. This deforms a small patch of the cell membrane and curves it inwards. The clathrin molecules coat this pocket as it grows in size, until it engulfs the cargo. The pocket quickly pinches off from the membrane to form a bubble-like structure called a vesicle, which is brought into the cell. A family of proteins termed Muniscins were thought to be involved in the early stages of endocytosis and have to arrive at the membrane before the adaptor protein-2 complex and clathrin. But experiments to test this idea—that reduced, or ‘knocked-down’, the production of Muniscins—had given conflicting results. As such, it remained unclear how the small patches of membrane carrying cargo molecules are marked as being destined to become clathrin-coated vesicles. Now Umasankar et al. have studied the role that these proteins play in the early stages of endocytosis in human cells grown in a laboratory. A gene-editing approach was used to precisely disrupt a gene that codes for a Muniscin protein called FCHO2. Umasankar et al. observed that these ‘edited’ cells formed clathrin coats that were more irregular compared with those that form in normal cells. Nevertheless, clathrin-mediated vesicles still formed when this protein was absent, though the process of endocytosis was slower. Similar results were seen when Umasankar et al. used the same approach to disrupt the gene for a related protein called FCHO1 in the same cells. A short fragment of the Muniscin proteins, called the linker, was shown to bind to, and activate, the adaptor protein-2 complex. The linker then recruits this complex to the specific regions of the cell membrane where clathrin-coated vesicles will form. Several dozen other proteins also accumulate where clathrin pockets form; as such, one of the next challenges will be to investigate if this mechanism of locally activating the cargo-gathering machinery is common in living cells. DOI: http://dx.doi.org/10.7554/eLife.04137.002 |
| تدمد: | 2050-084X |
| URL الوصول: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::c7151abc2022f75065f302fff9e04ee7 https://pubmed.ncbi.nlm.nih.gov/25303365 |
| Rights: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....c7151abc2022f75065f302fff9e04ee7 |
| قاعدة البيانات: | OpenAIRE |
| تدمد: | 2050084X |
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