The Karyopherin Kap95 Regulates Nuclear Pore Complex Assembly into Intact Nuclear Envelopes In Vivo

Abstract

Nuclear pore complex (NPC) assembly in interphase cells requires that new NPCs insert into an intact nuclear envelope (NE). Our previous work identified the Ran GTPase as an essential component in this process. We proposed that Ran is required for targeting assembly factors to the cytoplasmic NE face via a novel, vesicular intermediate. Although the molecular target was not identified, Ran is known to function by modulating protein interactions for karyopherin (Kap) beta family members. Here we characterize loss-of-function Saccharomyces cerevisiae mutants in KAP95 with blocks in NPC assembly. Similar to defects in Ran cycle mutants, nuclear pore proteins are no longer localized properly to the NE in kap95 mutants. Also like Ran cycle mutants, the kap95-E126K mutant displayed enhanced lethality with nic96 and nup170 mutants. Thus, Kap95 and Ran are likely functioning at the same stage in assembly. However, although Ran cycle mutants accumulate small cytoplasmic vesicles, cells depleted of Kap95 accumulated long stretches of cytoplasmic membranes and had highly distorted NEs. We conclude that Kap95 serves as a key regulator of NPC assembly into intact NEs. Furthermore, both Kap95 and Ran may provide spatial cues necessary for targeting of vesicular intermediates in de novo NPC assembly.

A dominant-negative form of POM121 binds chromatin and disrupts the two separate modes of nuclear pore assembly

“…NPCs also form at a separate stage of the metazoan cell cycle, by insertion into the intact NE during interphase (D'Angelo et al, 2006). This second mode of NPC formation enables metazoan cells to double NPC numbers during interphase (Maul et al, 1972) and more closely resembles the assembly pathway of lower eukaryotes, which undergo a closed mitosis (Fernandez-Martinez and Rout, 2009;Ryan et al, 2007).…”

“…The mechanistic details and sequence of events in NPC assembly have been a matter of controversy and debate. Although NPC biogenesis is often described as a self assembly process, there is also evidence for the involvement of additional assembly factors, such as importinb (Harel et al, 2003a;Ryan et al, 2007;Walther et al, 2003). The Nup107-160 complex is known to bind to chromatin through the adaptor protein ELYS and form the initial seeding sites, or 'prepores', for postmitotic NPC assembly (Franz et al, 2007;Gillespie et al, 2007;Rasala et al, 2006;Rasala et al, 2008;Rotem et al, 2009), but subsequent steps remain obscure.…”

Integral membrane proteins Brr6 and Apq12 link assembly of the nuclear pore complex to lipid homeostasis in the endoplasmic reticulum

“…These include components of the Ran GTPase system (Ryan and Wente, 2002), whose role in coordinating assembly and disassembly of karyopherin-cargo complexes during nucleocytoplasmic transport has been well studied (for reviews, see Conti et al, 2006;Cook et al, 2007;Stewart, 2007;Terry et al, 2007). Cells carrying specific mutant alleles affecting these proteins showed mislocalization of nucleoporins as well as accumulation of nucleoporin-containing cytoplasmic vesicles (Ryan and Wente, 2002;Ryan et al, 2003;Ryan et al, 2007). Proteins involved in endoplasmic reticulum (ER) to Golgi trafficking, including components of the COPII coat, have also been implicated in NPC assembly (Ryan and Wente, 2002).…”

NEP-A and NEP-B both contribute to nuclear pore formation in Xenopus eggs and oocytes

“…fusion of the vesicles into an ER-like network, enclosure of the chromatin and then NPC formation and NE expansion (Mattaj, 2004). In vitro and in vivo fusion events are regulated by the small GTPase Ran and importin ␤ (Hetzer et al, 2002;Zhang and Clarke, 2001;Walther et al, 2003a;Harel et al, 2003;Ryan et al, 2007). In vitro, fusion occurs between two distinct NEP types: NEP-B, which binds initially to chromatin; and NEP-A, which is required for fusion (Vigers and Lohka, 1991;Drummond et al, 1999).…”