Nuclear pore targeting of the yeast Pom33 nucleoporin depends on karyopherin- and lipid-binding

Abstract

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), required for proper NPC distribution and assembly. To characterize Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123. In silico analysis predicted the presence of two amphipathic α-helices within Pom33-CTD. Circular dichroism and liposome co-flotation assays showed that this domain is able to fold into α-helices in the presence of liposomes and preferentially binds to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, this domain behaves as a Kap123-dependent nuclear localization signal (NLS). While deletion of Pom33 C-terminal domain (Pom33ΔCTD-GFP) impairs Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Quantitative proteomics reveal proteins enriched in tubular endoplasmic reticulum of Saccharomyces cerevisiae

“…Furthermore, the membranes surrounding the nuclear pore have high curvature similar to that of ER tubules (Mészáros et al, 2015). POM33 (TU:TO 3.81; TU:BL 3.79) may provide additional linkage between nucleus biogenesis and ER tubules, as it has also been implicated in the nuclear pore complex distribution and NE remodeling (Casey et al, 2015; Chadrin et al, 2010; Floch et al, 2015). The localization of POM33 and its homologous proteins (PER33, Tts1, and TMEM33) can be explained by the presence of multiple TMHs and APHs and their interactions with known tubule proteins, including Rtn1p, Sey1p, and Lnp1p (Casey et al, 2015; Chadrin et al, 2010; Zhang and Oliferenko, 2014).…”

Yeast Integral Membrane Proteins Apq12, Brl1, and Brr6 Form a Complex Important for Regulation of Membrane Homeostasis and Nuclear Pore Complex Biogenesis

“…All other integral membrane nucleoporins are not essential, but generally only a single one can be absent. It has also been shown that targeting of Pom33, an integral membrane nucleoporin, to the NPC requires its amphipathic helices, which preferentially bind to highly curved membranes (12). Also implicated in NPC biogenesis are the reticulons, which are able to induce membrane curvature and have been shown to be important for the formation of tubule structures of the ER (40).…”

“…Additionally, many NPC proteins, including yeast Nup85, Nup120, and Nup133, contain amphipathic helical ALPS (ArfGAP1 lipid-packing sensor) motifs that sense lipid packaging at the curved membranes of the POM (11). Thus, a complex array of lipid-protein interactions is thought to stabilize the highly curved pore membranes, but the roles of these interactions in membrane fusion and curvature are not fully understood (12,13).…”

Nup153 Recruits the Nup107-160 Complex to the Inner Nuclear Membrane for Interphasic Nuclear Pore Complex Assembly

“…Rapid transportin-dependent import of Nup153 might prevent its membrane association outside of the nucleus. We wondered whether transportin interaction could also directly affect Nup153 membrane binding similar to Kap123, which regulates membrane interaction of the spindle body component Nbp1p and the C-terminal domain of the transmembrane nucleoporin Pom33p in yeast (Floch et al, 2015;Kupke et al, 2011). To test this, the N terminus of Nup153 was employed in liposome flotation assays after pre-incubation with transportin.…”