PubMed · 9827796
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B Pishvaee, G S Payne. 1998-11-13. Clathrin coats--threads laid bare.. https://doi.org/10.1016/s0092-8674(00)81611-6
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Rab14 localizes to the Golgi/TGN and to early endosomes, but its biological function remains unclear. By structural modeling, we identified Rab14-specific residues and established a close relationship between the Rab2/Rab4/Rab14, Rab11/25 and Rab39 sub-groups within the Rab protein family. By quantitative confocal microscopy and by density centrifugation we show that Rab14 is part of the early endosomal AP-1 microdomain. Overexpression of a dominant-negative Rab14 GTP-binding mutant that solely localizes to the Golgi donor compartment accelerated EGF degradation. We suggest that the AP-1 microdomain represents the interconnecting compartment in which Rab14 vesicles cycle between early endosomes and the Golgi cisternae.
Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.
In HeLa cells depleted of adaptor protein 2 complex (AP2) by small interfering RNA (siRNA) to the mu2 or alpha subunit or by transient overexpression of an AP2 sequestering mutant of Eps15, endocytosis of the transferrin receptor (TfR) was strongly inhibited. However, epidermal growth factor (EGF)-induced endocytosis of the EGF receptor (EGFR) was inhibited only in cells where the alpha subunit had been knocked down. By immunoelectron microscopy, we found that in AP2-depleted cells, the number of clathrin-coated pits was strongly reduced. When such cells were incubated with EGF, new coated pits were formed. These contained EGF, EGFR, clathrin, and Grb2 but not the TfR. The induced coated pits contained the alpha subunit, but labeling density was reduced compared to control cells. Induction of clathrin-coated pits required EGFR kinase activity. Overexpression of Grb2 with inactivating point mutations in N- or C-terminal SH3 domains or in both SH3 domains inhibited EGF-induced formation of coated pits efficiently, even though Grb2 SH3 mutations did not block activation of mitogen-activated protein kinase (MAPK) or phosphatidylinositol 3-kinase (PI3K). Our data demonstrate that EGFR-induced signaling and Grb2 are essential for formation of clathrin-coated pits accommodating the EGFR, while activation of MAPK and PI3K is not required.