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Published 15 October 2001. doi:10.1083/jcb.200104085
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© The Rockefeller University Press, 0021-9525/2001/10/291 $5.00
The Journal of Cell Biology, Volume 155, Number 2, October 15, 2001 291-300

Article

 Clathrin exchange during clathrin-mediated endocytosis

Xufeng Wu, Xiaohong Zhao, Lauren Baylor, Shivani Kaushal, Evan Eisenberg and Lois E. Greene

Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

Address correspondence to Lois Greene, Laboratory of Cell Biology, NHLBI, 50 South Dr., Rm. 2537 MSC 8017, Bethesda, MD 20892-8017. Tel.: (301) 496-1228. Fax: (301) 402-1519. E-mail: greenel{at}helix.nih.gov

During clathrin-mediated endocytosis, clathrin-coated pits invaginate to form clathrin-coated vesicles (CVs). Since clathrin-coated pits are planar structures, whereas CVs are spherical, there must be a structural rearrangement of clathrin as invagination occurs. This could occur through simple addition of clathrin triskelions to the edges of growing clathrin-coated pits with very little exchange occurring between clathrin in the pits and free clathrin in the cytosol, or it could occur through large scale exchange of free and bound clathrin. In the present study, we investigated this question by studying clathrin exchange both in vitro and in vivo. We found that in vitro clathrin in CVs and clathrin baskets do not exchange with free clathrin even in the presence of Hsc70 and ATP where partial uncoating occurs. However, surprisingly FRAP studies on clathrin-coated pits labeled with green fluorescent protein–clathrin light chains in HeLa cells show that even when endocytosis is blocked by expression of a dynamin mutant or depletion of cholesterol from the membrane, replacement of photobleached clathrin in coated pits on the membrane occurs at almost the same rate and magnitude as when endocytosis is occurring. Furthermore, very little of this replacement is due to dissolution of old pits and reformation of new ones; rather, it is caused by a rapid ATP-dependent exchange of clathrin in the pits with free clathrin in the cytosol. On the other hand, consistent with the in vitro data both potassium depletion and hypertonic sucrose, which have been reported to transform clathrin-coated pits into clathrin cages just below the surface of the plasma membrane, not only block endocytosis but also block exchange of clathrin. Taken together, these data show that ATP-dependent exchange of free and bound clathrin is a fundamental property of clathrin-coated pits, but not clathrin baskets, and may be involved in a structural rearrangement of clathrin as clathrin-coated pits invaginate.

Key Words: clathrin; exchange; FRAP; endocytosis; Hsc70


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