The TREX-2 complex is central to the integration of transcription and mRNA nuclear export (Rodriguez-Navarro et al., 2004; Masuda et al., 2005; Aguilera, 2005
), and the CID region of Sac3 and its associated binding to Sus1 and Cdc31 is crucial to this function. We have determined the crystal structure of the Sac3CID
:Cdc31:Sus1 complex and show that Sac3CID
binds two Sus1 chains (Sus1A and Sus1B) and a single Cdc31 chain. Our results refine the boundaries of the CID region and indicate that Sac3 residues 723–805 are necessary and sufficient for binding both Sus1 and Cdc31. In the complex, Sac3CID
forms a gently undulating extended α helix, whereas Sus1 is constructed from an articulated α-helical hairpin in which five α helices are joined by flexible hinges that facilitate the molecule's wrapping around Sac3 like fingers gripping a rod. Both Sus1 and Cdc31 wrap intimately around the Sac3 helix. The crystal structure identified the precise residues in the interfaces between the CID domain and its three partner chains, and this information was employed to engineer Sac3 mutants in which the binding to each partner was compromised. Sac3 mutants in which binding to the Sus1A, Sus1B, or Cdc31 sites was ablated showed different growth and mRNA nuclear export defects. Overall, our data indicate that, within TREX-2, the Sac3CID
complex facilitates integration of components of the transcriptional and mRNA nuclear export functions of the gene expression pathway in yeast. Moreover, the existence of metazoan Sac3 homologs and the demonstration that the human analog GANP can bind Sus1 indicated that similar principles probably apply in metazoans.
It has previously been difficult to evaluate the precise contribution made to TREX-2 function by the interactions of Sus1 and Cdc31 with Sac3 because both Sus1 and Cdc31 also participate in other cellular functions. Thus, in addition to its role in mRNA export (Fischer et al., 2004
), Cdc31 is essential for spindle pole body duplication (Ivanovska and Rose, 2001; Kilmartin, 2003
) and also appears to stabilize the Kar1 kinesin (Hu and Chazin, 2003
). Sus1, however, is a component of both the TREX-2 and SAGA complexes and has been proposed to provide a link between them (Rodriguez-Navarro et al., 2004
). The Sac3 mutants in which the binding to each site was specifically inhibited (ΔSus1A, ΔSus1B, and ΔCdc31) were able to circumvent this difficulty and demonstrated that the binding of both Sus1 and Cdc31 to Sac3CID
was important for efficient mRNA nuclear export (). Moreover, although Cdc31 and Sus1 appear to act in concert, the severity of both the growth defect and the mRNA export defect was different for each site, with the Sus1B site deletion having the largest defect and the Sus1A site deletion having the least (). NPC localization of GFP-Sac3CID
fragments was only observed when Sus1B was augmented with either Sus1A or Cdc31 binding (), again consistent with a high degree of synergy between the individual chains.
The extended α-helical conformation of Sac3CID
is a striking feature of the crystal structure of the Sac3CID
:Sus1:Cdc31 complex (). In its interactions with the spindle pole component, Sfi1 (Li et al., 2006
), and the Kar1 kinesin (Hu and Chazin, 2003
), Cdc31 appears to stabilize an extended α helix, analogous to the role proposed for light chains in myosin (Trybus, 2007
). Isolated extended α helices are generally unstable, and so it is likely that both Cdc31 and Sus1 contribute to the function of Sac3 in the TREX-2 complex by stabilizing the 12.5 nm long, extended Sac3CID
α helix. Indeed, one reason two Sus1 chains are required may be that a single chain is insufficient to cover the extraordinarily long Sac3CID
helix. However, additional functions for the Sac3CID
complex are indicated by the differential effects on growth, mRNA export, and the genetic interactions with other export factors seen with deletion of the Sus1A, Sus1B, and Cdc31 binding sites of Sac3. Indeed, a major function of binding of both Sus1s to Sac3 appears to facilitate the NPC tethering of Sac3CID
() that is central to the function of the TREX-2 complex in confining actively transcribing genes to the nuclear periphery (Cabal et al., 2006; Chekanova et al., 2008
). Moreover, Sus1 is thought to functionally link TREX-2 to the SAGA complex (Rodriguez-Navarro et al., 2004; Cabal et al., 2006; Köhler et al., 2008
), although structural information about the interfaces involved has yet to be obtained. However, a large interface of Sus1 remains exposed after it has wrapped around Sac3, and this could bind SAGA components in the context of the complete complexes. Although further work will be required to identify the complete inventory of components that interact with the Sac3CID
complex, it clearly appears to function within TREX-2 to facilitate integration of interactions between the transcription and mRNA export components of the gene expression pathway in yeast.
It is presently not clear whether the binding of Sus1 and Cdc31 to Sac3CID
is constitutive or is regulated during the gene expression pathway. Because it is a calmodulin homolog, Cdc31 has the potential to be regulated by cellular Ca2+
levels. However, analogous with the results obtained with the Cdc31:Sfi1 interaction (Li et al., 2006
), we were unable to see any influence of Ca2+
concentration on the interaction of Cdc31 with Sac3 (Figure S5
). In vitro binding studies (Figure S6
) indicated that Sus1 bound to Sac3 with ~10 nM affinity, which would be consistent with its remaining constitutively attached, although this would not preclude its binding being regulated by posttranscriptional modification. There is, for example, a putative acetylation site at Sac3 Lys748 that could potentially alter the local helical conformation and thereby control Sus1 binding (Rodriguez-Navarro et al., 2004; Fischer et al., 2004
The articulated hairpin fold of Sus1 does not appear to have close parallels in the protein structure database. However, the fold is ideally suited to wrapping around the Sac3CID
α helix. The flexibility introduced by the hinges between successive helices functions like the joints in a finger, enabling the relatively rigid helical segments of Sus1 to grasp Sac3. In addition, this manner of interaction leaves the outer surface of Sus1 free to bind to other components of the gene expression pathway, such as NPCs and the SAGA complex. Sus1 appears to recognize a motif characterized by hydrophobic residues repeating with an approximately four-residue period that generates a hydrophobic helical stripe that winds around the Sac3CID
rod. Using the information obtained on the Sus1:Sac3 interface, we were able to identify an analogous region within the human Sac3 homolog GANP and verified that it indeed bound the human Sus1 homolog DC6/ENY2. Further support for the more general applicability of the information we have obtained in yeast was recently provided by the observation that centrin 2, a protein with homology to Cdc31, was also found to be associated with NPCs and to be involved in mRNA and protein export (Resendes et al., 2008
). Additionally, in Drosophila
, Sac3/Xmas-2 and Sus1/E(y)2 are part of the AMEX complex (orthologous to yeast TREX-2) that is anchored at NPCs and regulates mRNA export (Kurshakova et al., 2007a, 2007b
). These observations indicate the CID motif of Sac3 and its associated binding of Sus1 and Cdc31 is a conserved structural and functional feature in many eukaryotes.
In summary, we have determined the structure of the Sac3CID:Sus1:Cdc31 complex and engineered Sac3 mutants that interfere with the binding of each partner to TREX-2 to appreciate how they function in gene gating. By contributing to confining actively transcribing genes to the peripheral regions of the nucleus via its interactions with both the SAGA complex and NPCs, TREX-2 facilitates mRNA generation in the vicinity of the NPCs, increasing its local concentration at the nuclear entrance of the transport channel, which should increase the efficiency of export. Overall, our data strongly indicate that the TREX-2 Sac3CID complex participates at several levels in the yeast gene expression pathway where it functions to coordinate and integrate the contributions from individual components of the machinery to promote efficient mRNA nuclear export.