Molecular characterization of the SUMO-1 modification of RanGAP1 and its role in nuclear envelope association.
The mammalian guanosine triphosphate (GTP)ase-activating protein RanGAP1 is the first example of a protein covalently linked to the ubiquitin-related protein SUMO-1. Here we used peptide mapping, mass spectroscopy analysis, and mutagenesis to identify the nature of the link between RanGAP1 and SUMO-1. SUMO-1 is linked to RanGAP1 via glycine 97, indicating that the last 4 amino acids of this 101- amino acid protein are proteolytically removed 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 proteins in vivo. In contrast to most ubiquitinated proteins, only a single lysine residue (K526) in RanGAP1 can serve as the acceptor site 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 nuclear protein import. Sufficient information for modification and targeting resides in a 25-kD domain of RanGAP1. RanGAP1-SUMO-1 remains stably associated with the NE during many cycles of in vitro import. This indicates that removal of RanGAP1 from the NE is not a required element of nuclear protein import and suggests that the reversible modification of RanGAP1 may have a regulatory role.
Mahajan
R
R
Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Gerace
L
L
Melchior
F
F
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D020690:GTPase-Activating Proteins; D005998:Glycine / Q000737:chemistry; D008239:Lysine / Q000737:chemistry; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009685:Nuclear Envelope / Q000378:metabolism*; D025842:SUMO-1 Protein; D012441:Saccharomyces cerevisiae; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism
D002352:Carrier Proteins; D020690:GTPase-Activating Proteins; D025842:SUMO-1 Protein; D014452:Ubiquitins; D008239:Lysine; D005998:Glycine