Recruitment dynamics of GAK and auxilin to clathrin-coated pits during endocytosis
Author:
Lee Dong-won1, Wu Xufeng1, Eisenberg Evan1, Greene Lois E.1
Affiliation:
1. Laboratory of Cell Biology, NHLBI, NIH, Bethesda, MD 20892-0301, USA
Abstract
Cyclin G-associated kinase (GAK), the ubiquitous form of the neuronal-specific protein auxilin 1, is an essential cofactor for Hsc70-dependent uncoating of clathrin-coated vesicles. Total internal reflectance microscopy was used to determine the timing of GAK binding relative to dynamin and clathrin binding during invagination of clathrin-coated pits. Following transient recruitment of dynamin to the clathrin puncta, large amounts of GAK are transiently recruited. GAK and clathrin then disappear from the evanescent field as the pit invaginates from the plasma membrane and finally these proteins disappear from the epifluorescence field, probably as the clathrin is uncoated from the budded vesicles by Hsc70. The recruitment of GAK is dependent on its PTEN-like domain, which we found binds to phospholipids. This suggests that interaction with phospholipids is essential for recruitment of GAK and, in turn, Hsc70, but Hsc70 recruitment alone might not be sufficient to induce irreversible clathrin uncoating. When budding of clathrin-coated pits is inhibited by actin depolymerization, there is repeated flashing of GAK on the clathrin-coated pit but neither scission nor irreversible uncoating occur. Therefore, budding as well as synchronous recruitment of GAK might be required for irreversible clathrin uncoating.
Publisher
The Company of Biologists
Reference37 articles.
1. Bar-Zvi, D., Mosley, S. T. and Branton, D. (1988). In vivo phosphorylation of clathrin-coated vesicle proteins from rat reticulocytes. J. Biol. Chem.263, 4408-4415. 2. Campbell, R. E., Tour, O., Palmer, A. E., Steinbach, P. A., Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2002). A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. USA99, 7877-7882. 3. Cremona, O., Di Paolo, G., Wenk, M. R., Luthi, A., Kim, W. T., Takei, K., Daniell, L., Nemoto, Y., Shears, S. B., Flavell, R. A. et al. (1999). Essential role of phosphoinositide metabolism in synaptic vesicle recycling. Cell99, 179-188. 4. Damke, H., Gossen, M., Freundlieb, S., Bujard, H. and Schmid, S. L. (1995). Tightly regulated and inducible expression of dominant interfering dynamin mutant in stably transformed HeLa cells. Meth. Enzymol.257, 209-220. 5. Ehrlich, M., Boll, W., Van Oijen, A., Hariharan, R., Chandran, K., Nibert, M. L. and Kirchhausen, T. (2004). Endocytosis by random initiation and stabilization of clathrin-coated pits. Cell118, 591-605.
Cited by
104 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献
|
|