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Clathrin exchange during clathrin-mediated endocytosis.

Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE - J. Cell Biol. (2001)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

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Model of clathrin exchange in vivo. See text for details.
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fig10: Model of clathrin exchange in vivo. See text for details.

Mentions: Since exchange is constantly occurring, the actual uncoating step may only involve a small change in the rates of clathrin attachment to and detachment from the membrane, particularly if either attachment or detachment is a cooperative process. Thus, as shown in the model in Fig. 10 Hsc70 may not only be involved in the uncoating of CVs after they pinch off from the membrane but may also be involved in the detachment of clathrin from clathrin-coated pits during earlier steps in endocytosis and perhaps in the rebinding of clathrin as well. In support of this view, our biochemical experiments suggest that Hsc70 is involved in both chaperoning dissociated clathrin and in the rebinding of clathrin to the membrane (Jiang et al., 2000). In addition, when auxilin RNAi was used to block formation of auxilin in C. elegans not only was clathrin-mediated endocytosis blocked, but in addition FRAP studies showed that in contrast to what occurs in WT C. elegans replacement of clathrin after photobleaching no longer occurred in the coelomocytes of RNAi worms (Greener et al., 2001). Although this could be due to clathrin-mediated endocytosis being blocked in the RNAi worms, it might also be due to clathrin exchange in clathrin-coated pits being blocked in the absence of auxilin, that is, in the absence of Hsc70 action.


Clathrin exchange during clathrin-mediated endocytosis.

Wu X, Zhao X, Baylor L, Kaushal S, Eisenberg E, Greene LE - J. Cell Biol. (2001)

Model of clathrin exchange in vivo. See text for details.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198830&req=5

fig10: Model of clathrin exchange in vivo. See text for details.
Mentions: Since exchange is constantly occurring, the actual uncoating step may only involve a small change in the rates of clathrin attachment to and detachment from the membrane, particularly if either attachment or detachment is a cooperative process. Thus, as shown in the model in Fig. 10 Hsc70 may not only be involved in the uncoating of CVs after they pinch off from the membrane but may also be involved in the detachment of clathrin from clathrin-coated pits during earlier steps in endocytosis and perhaps in the rebinding of clathrin as well. In support of this view, our biochemical experiments suggest that Hsc70 is involved in both chaperoning dissociated clathrin and in the rebinding of clathrin to the membrane (Jiang et al., 2000). In addition, when auxilin RNAi was used to block formation of auxilin in C. elegans not only was clathrin-mediated endocytosis blocked, but in addition FRAP studies showed that in contrast to what occurs in WT C. elegans replacement of clathrin after photobleaching no longer occurred in the coelomocytes of RNAi worms (Greener et al., 2001). Although this could be due to clathrin-mediated endocytosis being blocked in the RNAi worms, it might also be due to clathrin exchange in clathrin-coated pits being blocked in the absence of auxilin, that is, in the absence of Hsc70 action.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus