Posttranslational modification with ubiquitin-related proteins of the SUMO (small ubiquitin-related modifier) family is an essential mechanism in most eukaryotes. It regulates fate and function of many target proteins by changing their interactions with other macromolecules (reviewed in Hay, 2005
; Geiss-Friedlander and Melchior, 2007
; Wilkinson and Henley, 2010
). Covalent attachment of SUMO requires an enzymatic cascade consisting of a single E1-activating enzyme (see below), a single E2-conjugating enzyme (Ubc9), and one of several E3 ligases that facilitate transfer of SUMO from Ubc9 to the substrate. Specific SUMO isopeptidases make this modification reversible and highly dynamic (reviewed in Kim and Baek, 2009
). The majority of known SUMO targets are nuclear proteins, and, consistent with this, most of the known SUMO enzymes are enriched in this compartment.
The SUMO E1-activating enzyme is a heterodimer consisting of the two subunits: Aos1 (SAE1) and Uba2 (SAE2) (Dohmen et al.
; Johnson et al.
; Desterro et al.
; Gong et al.
; Okuma et al.
). Aos1 resembles the N-terminal, and Uba2 the C-terminal, part of the ubiquitin E1 enzyme (Lois and Lima, 2005
; Schulman and Harper, 2009
). In addition, Uba2 contains a C-terminal extension of ~80 amino acids. This region contains a cluster of basic amino acids reminiscent of a classical nuclear localization signal (NLS), and its deletion in Saccharomyces cerevisiae
leads to cytoplasmic mislocalization (Dohmen et al.
). Although S. cerevisiae
Uba2) is essential, partial or complete deletion of its C-terminal extension has only minor or no growth defects (Dohmen et al.
; del Olmo et al.
; Lois and Lima, 2005
). Consistent with this, SUMOylation patterns are largely unchanged (Lois and Lima, 2005
). One possible explanation for this finding is that sufficient levels of Uba2 still reach the nuclear compartment, for example, in association with Aos1. This is consistent with the finding that some Uba2 was still present in the yeast nuclei (Dohmen et al.
). An NLS in Aos1, however, has not been described, and it is unknown whether the SUMO E1 enzyme heterodimer assembles before or after nuclear import. To gain insights into these questions, we decided to characterize the nuclear import of mammalian SUMO E1 subunits and of the holo-enzyme.
Translocation of proteins through nuclear pore complexes (NPCs) is mediated by import and export receptors and requires the GTPase Ran (reviewed in Mattaj and Englmeier, 1998
; Fried and Kutay, 2003
; Weis, 2003
; Pemberton and Paschal, 2005
; Conti et al.
; Stewart, 2007
). Whereas nuclear import via classical NLSs (one or two short clusters of basic amino acids; first identified in SV40 large T antigen [Kalderon et al.
] and in nucleoplasmin [Robbins et al.
], respectively) is mediated by importin β together with the adaptor protein importin α (Adam and Gerace, 1991
; Gorlich et al.
; Moroianu et al.
; Weis et al.
), cargos bearing a nonclassical NLS (e.g., an M9 [Siomi and Dreyfuss, 1995
], BIB [Jäkel and Gorlich, 1998
], or RS domain [Kataoka et al.
; Lai et al.
]) can directly interact with distinct members of the importin β family.
Here we show that Aos1 and Uba2 have distinct functional NLSs and demonstrate that the Uba2 NLS is required for import of the holo-enzyme. Importin α/β functions as the main transport receptor for both individual subunits and the assembled E1 enzyme.