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.
Zhu
Shanshan
S
Johns Hopkins University, Bloomberg School of Public Health, Department of Biochemistry and Molecular Biology, 615 North Wolfe Street, Baltimore, MD 21205, USA.
Zhang
Hong
H
Matunis
Michael J
MJ
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