In this study, we describe a novel connection for the ER proteins Rtn1 and Yop1 with nuclear pore and NPC biogenesis. Previous studies have restricted Rtn1 and Yop1 function to the tubular ER with no known roles at the NPC reported (Geng et al., 2005
; De Craene et al., 2006
; Voeltz et al., 2006
; Shibata et al., 2008
). Several lines of evidence support our conclusions. First, we observe perturbations in Rtn1-GFP localization in the NPC assembly mutant prp20-G282S
. Second, Rtn1-GFP appears to concentrate at NPC clusters in a nup133Δ
mutant. Third, the lack of Rtn1 and Yop1 results in clusters of NPC-like structures in localized regions of the NE. Fourth, our genetic results reveal a tight link between Rtn1/Yop1 function at the NE with the Poms, the linker Nups, and the yNup84 subcomplex. Finally, using Xenopus
in vitro assays for de novo NPC assembly, we find that anti-Rtn4a antibodies specifically inhibit pore biogenesis. We conclude that a combination of RTN, Yop1/DP1, and Poms is required for the formation of nuclear pores and/or the insertion of NPCs into the pore. We predict that these membrane-bending proteins influence the stability of the pore membrane during NPC biogenesis. These results directly impact current models for de novo pore formation and ER/NE membrane dynamics.
Our yeast genetic experiments uncover separable roles for different membrane proteins at the NE/NPC. Strikingly, Ndc1 is not sufficient for growth or, presumably, NPC assembly. This is based on the lethality of the rtn1Δ yop1Δ pom34Δ pom152Δ
mutant. Furthermore, we find that the rtn1Δ yop1Δ
double mutant has specific genetic interactions with genes encoding Nups in the yNup84 subcomplex and in the linker Nup subcomplex. In contrast, strong connections to the inner ring Nups are not detected. In regard to ER/NE proteins, a specialized NE and/or nuclear pore lipid composition and membrane fluidity might also be required (Schneiter et al., 1996
; Scarcelli et al., 2007
). However, the reported abnormalities in NE/NPC morphology for mutants in genes encoding other NE-associated integral membrane proteins, including Acc1, Apq12, Brr6, Spo7, and Nem1 (Schneiter et al., 1996
; Siniossoglou et al., 1998
; de Bruyn Kops and Guthrie, 2001
; Scarcelli et al., 2007
), are structurally distinct from those in the rtn1Δ yop1Δ
mutant. Overall, our genetic results indicate that the Rtn1 and Yop1 role in nuclear pore biogenesis is separate from that for Apq12 and Brr6. Moreover, based on the genetic interactions with the spo7Δ
mutants, we speculate that Rtn1 and Yop1 have separable functional roles in tubular ER maintenance and in NE pore formation.
The RTNs and Yop1/DP1 exert their effects on tubular ER morphology through influencing membrane curvature and fusion events (De Craene et al., 2006
; Voeltz et al., 2006
; Hu et al., 2008
; Shibata et al., 2008
). Yeast (y)Rtn1 (or vRtn4a) interacts with yYop1 (vDP1), and both are membrane proteins with a similar predicted hairpin topology (Voeltz et al., 2006
). It is proposed that their respective extended hydrophobic domains insert only into the outer ER membrane leaflet as oligomers. Their unusual wedge-shaped membrane spans presumably displace lipid head groups to induce and stabilize a highly curved membrane structure in a manner proposed by the classical bilayer couple hypothesis (Sheetz et al., 1976
; Voeltz et al., 2006
There are several models for how these membrane proteins mediate nuclear pore formation. A role strictly involving ER tubules formed by Rtn1 and Yop1 could underlie the NPC connections. Recent reports link the functions of metazoan RTNs in tubular ER maintenance to both premitotic NE disassembly and NE postmitotic reassembly (Anderson and Hetzer, 2007
; Audhya et al., 2007
). In addition, vRtn4a is localized to highly curved membrane junctions between cytoplasmic membranes and the ONM (Kiseleva et al., 2007
). The Rtn1/Yop1 ER tubules might connect to the NE and trigger transient ONM curvature. The tubular ER might also be required for localized delivery of NPC precursors such as POMs to the NE. However, strikingly, the Xenopus
in vitro assays () demonstrate that new NPC and pore biogenesis is inhibited by anti-Rtn4a antibodies at times when tubular ER connections to the NE are intact. This further supports our conclusion that RTNs play roles in NPC formation that are distinct from their function in maintaining tubular ER.
We propose that the RTNs and/or Yop1/DP1 functions directly at the NE to facilitate NPC biogenesis. A fraction of yeast Rtn1 has been observed at the NE by both yRtn1-GFP imaging (; Geng et al., 2005
) and vRtn4a immuno-EM (Kiseleva et al., 2007
). Our genetic interaction results indicate that Rtn1 and Yop1 are specifically linked to discrete Nup and Pom subcomplexes and to only a subset of specific ER/NE proteins (). Thus, there is no pleiotropic defect in the delivery of integral membrane proteins to the NE/NPC in rtn1Δ yop1Δ
mutants. Furthermore, we find that the lethal pom34Δ nup59Δ
mutant is more efficiently rescued by the NE-enriched rtn1-K48I
mutant, which also has decreased oligomerization and greater membrane mobility (Shibata et al., 2008
). Collectively, with the Xenopus
in vitro assembly data, we propose that yeast Rtn1 (vRtn4a) and Yop1 function in the NE to stabilize the highly curved nuclear pore membranes.
Fusion of the INM and ONM is thought to be facilitated largely by the Poms; however, RTNs and/or Yop1/DP1 might independently be involved in localized bending of the ONM or INM to assist fusion. Alternatively, RTNs and/or Yop1/DP1 might be recruited after fusion to the nascent highly curved membrane, with their topological insertion into only the outer leaflet of the pore membrane, resulting in a stabilized nascent pore. In either of these scenarios, a physical interaction between the RTNs and/or Yop1/DP1 with NPC components is not necessarily required. Of note, a genome-wide split ubiquitin two-hybrid screen has reported an uncharacterized Yop1–Pom34 interaction (Miller et al., 2005
). The colocalization of yeast Rtn1 and Yop1 with NPC clusters in nup133Δ
cells could reflect a physical Pom–Nup interaction and a role at preexisting NPCs. However, proteomic studies have not reported the coisolation of Rtn1 or Yop1 with wild-type detergent-solubilized yeast NPC or Nup subcomplexes (Rout et al., 2000
; Cronshaw et al., 2002
; Alber et al., 2007a
). The colocalization with nup133Δ
NPC clusters might only reflect recruitment to a site with an increased density of defectively assembled NPCs that retain Rtn1 and Yop1. The insertion of RTNs and/or Yop1/DP1 in the outer leaflet of the pore membrane and localization of Pom–Nups to these membranes could be mutually exclusive sterically. Our future work will investigate whether RTN or Yop1/DP1 physically interacts with NPC components or whether they simply function to transiently maintain/induce membrane curvature during critical early steps of pore formation. In this latter model, subsequent incorporation of additional Nups–Poms would terminally stabilize pore structure and remove the need for the RTNs at mature NPCs.
Multiple members of the yNup84 subcomplex contain predicted amphipathic α-helix motifs implicated in sensing membrane curvature (Drin et al., 2007
) or have structural homology to clathrin and coatomer protein I (COPI)/COPII vesicle coat proteins (Siniossoglou et al., 1996
; Devos et al., 2004
; Mans et al., 2004
; Hsia et al., 2007
). In vesicle budding, initiation of membrane curvature is promoted by accessory proteins that interact with the outer bilayer leaflet via amphiphatic α helices, α-solenoid, and β-propeller motifs, and/or Bin–amphiphysin–Rvs (BAR) domains (Farsad and De Camilli, 2003
; Stagg et al., 2007
). Subsequent recruitment of the respective COP or clathrin coat proteins is thought to maintain the highly curved vesicle membrane. Thus, in a similar manner, the yNup84 and vNup107–160 subcomplex components are prime candidates for sustaining the mature NPC's curved pore membrane after the proposed initial RTN and/or Yop1/DP1 association with the pore. If RTNs and/or Yop1/DP1 is absent in mature NPCs, this could also facilitate NPC disassembly and allow for an unstable pore membrane to be rapidly resolved to an intact NE in interphase or during mitosis to ER tubules.
The formation of pores spanning the INM and ONM remains a poorly understood membrane fusion process. Collectively, our data strongly implicate RTNs and/or Yop1/DP1 as membrane proteins involved in nuclear pore formation and/or stability. The RTNs and Yop1/DP1 could function as underlying modulators of nuclear pore biogenesis, increasing assembly efficiency by stabilizing early pore assembly events and potentially coordinating the actions of Poms and the membrane-coating yNup84/vNup107–160 subcomplex. This work also highlights the overall dynamics of membrane systems in the cell and reveals further interconnections between the NE and tubular ER.