| Summary: | 碩士 === 臺灣大學 === 微生物學研究所 === 98 === The GTPase activating protein 1 of Ran (RanGAP1), which plays a vital role in nucleocytoplasmic transport, is the first modification substrate for small-ubiquitin-like-modifier (SUMO) identified. SUMOylation targets RanGAP1 to nuclear rim and has been suggested as an important regulation for its nucleocytoplasmic transport function. Nevertheless, other post-translational modification(s) on RanGAP1 and their influences on cellular function(s) of RanGAP1 remained largely unknown. In this thesis, we demonstrated that intracellular Ca2+ influx induces limited proteolysis on RanGAP1 and thereby potentially modulates nucleocytoplasmic transport of substrate proteins. First, this proteolytic event could be attenuated by Ca2+ chelators, indicating the involvement of Ca2+ in RanGAP1 proteolysis. Using biochemical, pharmacological and genetic approaches, we showed that calpain 2, a Ca2+-dependent cysteine protease, is mainly responsible for this Ca2+-mediated RanGAP1 cleavage. Through in vitro calpain 2 cleavage assay with recombinant RanGAP1proteins, and followed with the N-terminal sequencing, two major calpain 2-cleaved sites were identified: (i) Thr60-Val61 on N’-terminal leucine-rich-repeat and (ii) Ser427-Ser428 between acidic domain and C’-terminal tail. Further studies indicated that calpain 2-mediated proteolytic cleavages of RanGAP1 potentially caused its translocation from nuclear rim to cytoplasm, probably through proteolytic removal of its C’-terminus where SUMOylation occurred. Similarly, the N’-terminal truncation of RanGAP1 by calpain 2 seemed to delocalize RanGAP1 to nucleus possibly due to lost of domains containing nuclear export sequence (NES). In sum, our results suggest a potential proteolytic modification of RanGAP1 via calpain 2 and its influences on cellular locations of RanGAP1 and nucleocytoplasmic transport of reporter substrate proteins. The implications of our study for the RanGAP1-mediated biological functions therefore need further investigation.
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