Nuclear Pore Complex Assembly

Eukaryotes require continual exchange of macromolecules between the nucleus and the cytoplasm. Nuclear pore complexes (NPCs) are large, proteinaceous structures that span the fused inner and outer nuclear membranes to form the passageway. Multiple copies of ~30 different proteins, collectively termed nucleoporins, comprise the mature NPC. Its overall structure is characterized by 8-fold rotational symmetry. A combination of spoke- and ring-like substructures forms a core to which cytoplasmic fibrils and a nuclear basket attach. Changes in growth conditions, developmental state, signaling cascades, cell cycle, and viral infection affect which proteins and RNAs are trafficked between the nucleus and the cytoplasm; however, all movement occurs through NPCs. Therefore, elucidating the molecular mechanism of NPC assembly and determining the interactions required for its structural integrity are the long-term research goals in the Ryan lab.

The lab takes advantage of the yeast Saccharomyces cerevisiae to focus on the mechanism of NPC assembly. As a postdoc, Dr. Ryan conducted a genetic screen to identify factors required for NPC assembly using GFP-reporters in live cells. Using this approach, a large number of n uclear p ore complex a ssembly (npa) mutant were isolated. Current research in the Ryan lab has two major objectives:

1. Delineate the function of the Ran cycle in NPC assembly
Ran is a small GTPase that cycles between a GTP and GDP bound form to regulate many nuclear processes. All 4 components of the Ran cycle were isolated in the npa screen. Characterization of these mutants revealed membrane defects and the accumulation of nucleoporin containing vesicles in the cytoplasm. The accumulation of such vesicles in these npa mutants suggests that NPC assembly involves a Ran-mediated vesicular fusion event at the outer nuclear envelope. In this model of NPC assembly, a subset of nucleoporins is first concentrated in vesicles (A). When the vesicles fuse with the outer nuclear membrane in a Ran-dependent manner (B), a critical, localized concentration of these nucleoporins triggers pore formation (C) and nucleates new NPC assembly (D and E).

To test the model, work is being done to characterize these vesicles. This includes biochemical approaches to purify vesicles and cell biological and genetic approaches to determine how vesicle-associated proteins contribute to NPC assembly. In addition, we are working to understand how Ran interacts with these vesicles to mediate vesicle fusion to the outer nuclear membrane.

2. Define additional steps in the NPC assembly pathway
There are events both upstream and downstream of the Ran cycle in the assembly pathway. Further cloning and characterization of mutants from the npa collection will continue to identify factors involved in other steps of NPC biogenesis and provide a platform from which to study these discrete events.

1. Liu, D, Wu, X, Summers, MD, Lee, A, Ryan, KJ, Braunagel, SC et al.. Truncated isoforms of Kap60 facilitate trafficking of Heh2 to the nuclear envelope. Traffic. 2010;11 (12):1506-18. doi: 10.1111/j.1600-0854.2010.01119.x. PubMed PMID:20846261 .
2. Titus, LC, Dawson, TR, Rexer, DJ, Ryan, KJ, Wente, SR. Members of the RSC chromatin-remodeling complex are required for maintaining proper nuclear envelope structure and pore complex localization. Mol. Biol. Cell. 2010;21 (6):1072-87. doi: 10.1091/mbc.e09-07-0615. PubMed PMID:20110349 PubMed Central PMC2836959.
3. Ryan, KJ, Zhou, Y, Wente, SR. The karyopherin Kap95 regulates nuclear pore complex assembly into intact nuclear envelopes in vivo. Mol. Biol. Cell. 2007;18 (3):886-98. doi: 10.1091/mbc.e06-06-0525. PubMed PMID:17182855PubMed Central PMC1805111.
4. Miao, M, Ryan, KJ, Wente, SR. The integral membrane protein Pom34p functionally links nucleoporin subcomplexes. Genetics. 2006;172 (3):1441-57. doi: 10.1534/genetics.105.052068. PubMed PMID:16361228PubMed Central PMC1456286.
5. Ryan, KJ, McCaffery, JM, Wente, SR. The Ran GTPase cycle is required for yeast nuclear pore complex assembly. J. Cell Biol. 2003;160 (7):1041-53. doi: 10.1083/jcb.200209116. PubMed PMID:12654904 PubMed Central PMC2172763.
6. Ryan, KJ, Wente, SR. Isolation and characterization of new Saccharomyces cerevisiae mutants perturbed in nuclear pore complex assembly. BMC Genet. 2002;3 :17. . PubMed PMID:12215173 PubMed Central PMC126250.
7. Ho, AK, Shen, TX, Ryan, KJ, Kiseleva, E, Levy, MA, Allen, TD et al.. Assembly and preferential localization of Nup116p on the cytoplasmic face of the nuclear pore complex by interaction with Nup82p. Mol. Cell. Biol. 2000;20 (15):5736-48. . PubMed PMID:10891509 PubMed Central PMC86051.
8. Ryan, KJ, Wente, SR. The nuclear pore complex: a protein machine bridging the nucleus and cytoplasm. Curr. Opin. Cell Biol. 2000;12 (3):361-71. . PubMed PMID:10801463 .