Targeted chemical disruption of clathrin function in living cells.
The accurate assignment of molecular roles in membrane traffic is frequently complicated by the lack of specific inhibitors that can work on rapid time scales. Such inhibition schemes would potentially avoid the complications arising from either compensatory gene expression or the complex downstream consequences of inhibition of an important protein over long periods (>12 h). Here, we developed a novel chemical tool to disrupt clathrin function in living cells. We engineered a cross-linkable form of clathrin by using an FK506-binding protein 12 (FKBP)-clathrin fusion protein that is specifically oligomerized upon addition of the cell-permeant cross-linker FK1012-A. This approach interrupts the normal assembly-disassembly cycle of clathrin lattices and results in a specific, rapid, and reversible approximately 70% inhibition of clathrin function. This approach should be applicable to a number of proteins that must go through an assembly-disassembly cycle for normal function.
Moskowitz
Howard S
HS
Department of Biochemistry, Weill Medical College of Cornell University, New York, New York 10021, USA.
Heuser
John
J
McGraw
Timothy E
TE
Ryan
Timothy A
TA
D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D016466:CHO Cells; D002966:Clathrin / Q000187:drug effects / Q000378:metabolism*; D003001:Cloning, Molecular; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D036681:Fluorescence Recovery After Photobleaching; D008853:Microscopy; D008854:Microscopy, Electron; D011485:Protein Binding; D016559:Tacrolimus / Q000031:analogs & derivatives* / Q000494:pharmacology*; D022021:Tacrolimus Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002966:Clathrin; C084260:FK 1012; D013844:Thiazoles; D053778:Thiazolidines; D022021:Tacrolimus Binding Proteins; C037067:latrunculin A; D016559:Tacrolimus