Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

doi:10.1083/jcb.140.2.259

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© The Rockefeller University Press, 0021-9525/1998//259 $5.00
The Journal of Cell Biology, Volume 140, Number 2, , 1998 259-270

Article

Molecular Characterization of the SUMO-1 Modification of RanGAP1 and Its Role in Nuclear Envelope Association

Rohit Mahajan, Larry Gerace, and Frauke Melchior

Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037

The mammalian guanosine triphosphate (GTP)ase-activating proteinRanGAP1 is the first example of a protein covalently linkedto the ubiquitin-related protein SUMO-1. Here we used peptidemapping, mass spectroscopy analysis, and mutagenesis to identifythe nature of the link between RanGAP1 and SUMO-1. SUMO-1 islinked to RanGAP1 via glycine 97, indicating that the last4 amino acids of this 101– amino acid protein are proteolyticallyremoved before its attachment to RanGAP1. Recombinant SUMO-1 lacking the last four amino acids is efficiently used for modification of RanGAP1 in vitro and of multiple unknown proteinsin vivo. In contrast to most ubiquitinated proteins, only asingle lysine residue (K526) in RanGAP1 can serve as the acceptorsite for modification by SUMO-1. Modification of RanGAP1 with SUMO-1 leads to association of RanGAP1 with the nuclear envelope(NE), where it was previously shown to be required for nuclearprotein import. Sufficient information for modification andtargeting resides in a 25-kD domain of RanGAP1. RanGAP1–SUMO-1remains stably associated with the NE during many cycles ofin vitro import. This indicates that removal of RanGAP1 fromthe NE is not a required element of nuclear protein import andsuggests that the reversible modification of RanGAP1 may havea regulatory role.

Abbreviations used in this paper: GST, glutathione-S-transferase; HA, hemagglutinin; NE, nuclear envelope; NPC, nuclear pore complex; RT, room temperature; wt, wild type.

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