Distinct domains in Ndc1 mediate its interaction with the Nup84 complex and the nuclear membrane

Abstract

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope and built from ∼30 different nucleoporins (Nups) in multiple copies, few are integral membrane proteins. One of these transmembrane nucleoporins, Ndc1, is thought to function in NPC assembly at the fused inner and outer nuclear membranes. Here, we show a direct interaction of Ndc1’s transmembrane domain with Nup120 and Nup133, members of the pore membrane coating Y-complex. We identify an amphipathic helix in Ndc1’s C-terminal domain binding highly curved liposomes. Upon overexpression, this amphipathic motif is toxic and dramatically alters the intracellular membrane organization in yeast. Ndc1’s amphipathic motif functionally interacts with related motifs in the C-terminus of the nucleoporins Nup53 and Nup59, important for pore membrane binding and interconnecting NPC modules. The essential function of Ndc1 can be suppressed by deleting the amphipathic helix from Nup53. Our data indicate that nuclear membrane and presumably NPC biogenesis depends on a balanced ratio between amphipathic motifs in diverse nucleoporins.

Puzzling out nuclear pore complex assembly

“…While a constant supply of Nups is needed to cope with the assembly of NPCs, whose number gradually doubles during each cell cycle in dividing yeasts [4] or human cells [5,22], the accumulation of orphan , that is, nonassembled, subunits can in turn be detrimental for cellular homeostasis. In this line, overexpression of nucleoporins reportedly leads to distinct phenotypic outcomes, likely reflecting their different biochemical properties and interaction partners: (a) NE deformation or aberrant membrane structures have been observed upon accumulation of full‐length or truncated membrane‐binding Nups such as vNup153 [23], yNup53 [24], yNup1, yNup60 [25], or yNdc1 [26]; (b) cytoplasmic foci trapping a subset of Nups have been detected upon overproduction of a fragment of the scaffold nucleoporin yNup170 [27]; (c) increased dosage of different FG‐Nups, for example, vNup62 [28] or yNup1 [29], leads to their accumulation within ectopic aggregates. In these situations, mislocalized subunits have not only the potential to interfere with NPC assembly and NE homeostasis, but could also directly impact nucleocytoplasmic transport by titrating NTRs, as suggested by the trapping of the karyopherin yKap60 within yNup1 foci [30], or the displacement of the mRNA export receptor yMex67 upon overexpression of yNup116 GLFG domain [31].…”