SUMO modification through rapamycin-mediated heterodimerization reveals a dual role for Ubc9 in targeting RanGAP1 to nuclear pore complexes.
SUMOs (small ubiquitin-related modifiers) are eukaryotic proteins that are covalently conjugated to other proteins and thereby regulate a wide range of important cellular processes. The molecular mechanisms by which SUMO modification influences the functions of most target proteins and cellular processes, however, remain poorly defined. A major obstacle to investigating the effects of SUMO modification is the availability of a system for selectively inducing the modification or demodification of an individual protein. To address this problem, we have developed a procedure using the rapamycin heterodimerizer system. This procedure involves co-expression of rapamycin-binding domain fusion proteins of SUMO and candidate SUMO substrates in living cells. Treating cells with rapamycin induces a tight association between SUMO and a single SUMO substrate, thereby allowing specific downstream effects to be analyzed. Using RanGAP1 as a model SUMO substrate, the heterodimerizer system was used to investigate the molecular mechanism by which SUMO modification targets RanGAP1 from the cytoplasm to nuclear pore complexes (NPCs). Our results revealed a dual role for Ubc9 in targeting RanGAP1 to NPCs: In addition to conjugating SUMO-1 to RanGAP1, Ubc9 is also required to form a stable ternary complex with SUMO-1 modified RanGAP1 and Nup358. As illustrated by our studies, the rapamycin heterodimerizer system represents a novel tool for studying the molecular effects of SUMO modification.
Johns Hopkins University, Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Shanshan
S
Zhu
Hong
H
Zhang
Michael J
MJ
Matunis
D019281:Dimerization; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism; D022022:Nuclear Pore / Q000187:drug effects / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000378:metabolism; D025842:SUMO-1 Protein / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*; D014162:Transfection; D044763:Ubiquitin-Conjugating Enzymes / Q000502:physiology*; D000098843:Ubiquitin-Conjugating Enzyme UBC9
D020690:GTPase-Activating Proteins; D018832:Molecular Chaperones; D028861:Nuclear Pore Complex Proteins; C092317:RANGAP1 protein, human; D025842:SUMO-1 Protein; C094493:ran-binding protein 2; D044763:Ubiquitin-Conjugating Enzymes; D000098843:Ubiquitin-Conjugating Enzyme UBC9; D008239:Lysine; D020123:Sirolimus