Now that most of the yeast nucleoporins have been cloned and characterized, the next goal is to put them into a higher order biochemical and structural context. Here, we report on the molecular and structural characterization of the yeast Nup84p complex. This NPC subassembly consists of six subunits, five of which are bona fide nucleoporins (Siniossoglou et al. 1996
). When the essential nucleoporins of this complex are mutated, inhibition of nuclear mRNA export and defects in the distribution of NPCs and in the organization of the nuclear membrane occur. In the case of Nup85p, a physical and genetic link to the mRNA export machinery could be shown (Segref et al. 1997
; Santos-Rosa et al. 1998
), whereas Nup84p genetically interacts with two novel nuclear/ER membrane proteins, Spo7p and Nem1p, required for formation of a spherical nucleus (Siniossoglou et al. 1998
). However, the role of the sixth member of the Nup84p complex, Sec13p, a bona fide subunit of the COPII coat complex, remained unclear. We now demonstrate that a pool of Sec13p functions as a nucleoporin. Several lines of evidence support a role for Sec13p at the nuclear pore. First, we found that a pool of Sec13p-GFP colocalizes with NPCs in a clustering nucleoporin mutant. Apparently, this reflects the same pool of Sec13p, which physically interacts with the Nup84p complex. Second, the distribution of the GFP-Nup49p, which serves as a nucleoporin reporter, is altered in several of the newly isolated ts sec13
mutants. Strikingly, thin sectioning EM of the sec13-3
, and sec13-34
cells shifted to the restrictive temperature reveals abnormalities within the nuclear membrane and an accumulation of intracellular membranes, which could be nuclear envelope–attached ER. In addition, NPC herniations arise within the nuclear envelope of sec13
ts mutants, typically found in other nucleoporin mutants such as nup116Δ
. This all shows that Sec13p is required for normal nuclear envelope and NPC biogenesis. Interestingly, another COPII mutant, sec23-1
, also exhibits defects in the distribution of the GFP-Nup49p nucleoporin reporter. Mutants blocking COPII function accumulate ER membranes at the nonpermissive temperature. Thus, it is possible that the abnormal NPC distribution in the sec23
mutant is due to the accumulation of nuclear pores into these ER membranes, before their incorporation into the nuclear membrane. Alternatively, there might be a more direct link between COPII vesicle budding from the ER and the biogenesis of nuclear pores. Third, two ts alleles, sec13-14
, are synthetically lethal with the nup85Δ
mutant at 32°C, showing that Nup85p and Sec13p perform overlapping or redundant functions at the NPC. Surprisingly, combined seh1
mutant alleles do not exhibit such a genetic interaction.
Our biochemical analysis revealed that Nup120p, Nup145p-C, and the COOH-terminal domain of Nup85p are essential for the assembly of a core complex in vivo and thereby may serve as an assembly core or structural scaffold of the complex. Evidently, Nup84p, Seh1p, and Sec13p reside more peripherally within the Nup84p complex. Strikingly, the three members of the core complex exhibit a strong link to the mRNA export machinery (Fabre et al. 1994
; Aitchison et al. 1995
; Goldstein et al. 1996
; Siniossoglou et al. 1996
; Teixeira et al. 1997
). Hence, it is conceivable that the Nup84p complex consists of a structural scaffold, which at the same time is involved in nuclear mRNA export (e.g., by interacting with mRNA export factors such as Mex67p and Mtr2p; see also Santos-Rosa et al. 1998
). In contrast, the more peripheral, dispensable members of the complex (Nup84p, Seh1p, Sec13p) control the distribution of NPCs within the nuclear membrane as well as nuclear envelope organization. In the case of the nonessential Seh1p, we could show that it directly binds to the NH2
-terminal domain of Nup85p as one subunit of the core complex; this interaction is stable up to high salt concentrations (Siniossoglou et al. 1996
), and a separate Nup85p–Seh1p heterodimeric complex is even detected in vivo (this study). The role of this heterodimeric Nup85p–Seh1p complex is presently unknown, but it could play a direct role in nucleocytoplasmic transport or represent a stable intermediate during NPC assembly.
The highly purified Nup84p complex exhibits a defined structure as revealed by EM of negatively stained as well as glycerol-sprayed/low-angle rotary metal-shadowed specimens. Although not appearing 100% homogeneous in the EM, >50% of the highly purified Nup84p complex exhibits a distinct Y-shaped or triskelion-like morphology with an overall particle diameter of ~25 nm. We have estimated that there are between 8 and 16 copies of the Nup84p complex per NPC (Lutzman, M., and E. Hurt, unpublished data). Hence, the Nup84p complex makes a significant contribution to the overall mass of the NPC. Because of its apparent twofold symmetry, one possibility is that in situ, the Nup84p complex may assemble into an octagonal ring-like array, for instance, via interaction of the Y-shaped arms of adjacent complexes. In such a model, the third arm of each Nup84p complex unit remains free, and thus could interact with other components of the NPC, for example with a cytoplasmic fibril, and/or with transport factors. Alternatively, the Nup84p complex may oligomerize so that all three arms of a Y-shaped molecule are engaged in binding to other Y-shaped Nup84p complex molecules, thereby forming a coat- or cage-like structure. Finally, we cannot exclude that individual Y-shaped Nup84p particles exist within the structural framework of the NPC. Further structural and biochemical analysis of the Nup84p complex and its organization into higher-order complexes, including identification of its interacting components at the NPC, should help to answer these open questions.
Given the finding that the central NPC framework is conserved from yeast to vertebrates (Yang et al. 1998
), it would be predicted that a homologue to the Nup84p complex might also exist in higher eukaryotes. Indeed, a recent study reported the identification of a mammalian NPC subcomplex containing the homologues of cleaved yeast Nup145p-C and Nup84p. Intriguingly, this complex also contains mammalian Sec13p and a Sec13p-related protein showing that the function of Sec13p at the nuclear pore has been evolutionarily conserved (Fontoura et al. 1999
). It is therefore conceivable that the overall structure of the NPC is built from distinct subcomplexes or structural modules, which have been conserved during eukaryotic evolution.
In summary, the Nup84p complex appears to be a paradigm for the structural and functional analysis of an NPC subcomplex or a structural module. Amenable to yeast genetics and cell biological methods in vivo, as well as to biochemical purification and EM in vitro, it may be possible to reconstruct its 3-D structure, and to fit, i.e., to position and orient it into a 3-D mass density map of the entire NPC. Last but not least, it may be possible to use the purified Nup84p complex as a seed to reconstitute step-by-step an NPC in vitro from its purified nucleoporins and/or distinct subcomplexes.