Quantitative analysis of nuclear pore complex organization in Schizosaccharomyces pombe.

The number, distribution, and composition of nuclear pore complexes (NPCs) in the nuclear envelope varies between cell types and changes during cellular differentiation and in disease. To understand how NPC density and organization are controlled, we analyzed the NPC number and distribution in the fission yeast Schizosaccharomyces pombe using structured illumination microscopy. The small size of yeast nuclei, genetic features of fungi, and our robust image analysis pipeline allowed us to study NPCs in intact nuclei under multiple conditions. Our data revealed that NPC density is maintained across a wide range of nuclear sizes. Regions of reduced NPC density are observed over the nucleolus and surrounding the spindle pole body (SPB). Lem2-mediated tethering of the centromeres to the SPB is required to maintain NPC exclusion near SPBs. These findings provide a quantitative understanding of NPC number and distribution in S. pombe and show that interactions between the centromere and the nuclear envelope influences local NPC distribution.

A perinuclear α-helix with amphipathic features in Brl1 promotes NPC assembly.

How nuclear pore complexes (NPCs) assemble in the intact nuclear envelope (NE) is only rudimentarily understood. Nucleoporins (Nups) accumulate at the inner nuclear membrane (INM) and deform this membrane toward the outer nuclear membrane (ONM), and eventually INM and ONM fuse by an unclear mechanism. In budding yeast, the integral membrane protein Brl1 that transiently associates with NPC assembly intermediates is involved in INM/ONM fusion during NPC assembly but leaving the molecular mechanism open. AlphaFold predictions indicate that Brl1-like proteins carry as common motifs an α-helix with amphipathic features (AαH) and a disulfide-stabilized, anti-parallel helix bundle (DAH) in the perinuclear space. Mutants with defective AαH (brl1F391E, brl1F391P, brl1L402E) impair the essential function of BRL1. Overexpression of brl1F391E promotes the formation of INM and ONM enclosed petal-like structures that carry Nups at their base, suggesting that they are derived from an NPC assembly attempt with failed INM/ONM fusion. Accordingly, brl1F391E expression triggers mislocalization of Nup159 and Nup42 and to a lesser extent Nsp1, which localize on the cytoplasmic face of the NPC. The DAH also contributes to the function of Brl1, and AαH has functions independent of DAH. We propose that AαH and DAH in Brl1 promote INM/ONM fusion during NPC assembly.

A short perinuclear amphipathic α-helix in Apq12 promotes nuclear pore complex biogenesis.

The integral membrane protein Apq12 is an important nuclear envelope (NE)/endoplasmic reticulum (ER) modulator that cooperates with the nuclear pore complex (NPC) biogenesis factors Brl1 and Brr6. How Apq12 executes these functions is unknown. Here, we identified a short amphipathic α-helix (AαH) in Apq12 that links the two transmembrane domains in the perinuclear space and has liposome-binding properties. Cells expressing an APQ12 (apq12-ah) version in which AαH is disrupted show NPC biogenesis and NE integrity defects, without impacting Apq12-ah topology or NE/ER localization. Overexpression of APQ12 but not apq12-ah triggers striking over-proliferation of the outer nuclear membrane (ONM)/ER and promotes accumulation of phosphatidic acid (PA) at the NE. Apq12 and Apq12-ah both associate with NPC biogenesis intermediates and removal of AαH increases both Brl1 levels and the interaction between Brl1 and Brr6. We conclude that the short amphipathic α-helix of Apq12 regulates the function of Brl1 and Brr6 and promotes PA accumulation at the NE possibly during NPC biogenesis.