Abstract
ABSTRACTPom152 is a transmembrane protein within the nuclear pore complex (NPC) of fungi that is important for NPC assembly and structure. Pom152 is comprised of a short amino-terminal region that remains on the cytosolic side of the nuclear envelope (NE) and interacts with NPC proteins, a transmembrane domain, and a large, glycosylated carboxy-terminal domain within the NE lumen. Here we show that the N-terminal 200 amino acids of Pom152 that include only the amino-terminal and transmembrane regions are sufficient for localization to the NPC. Full-length, glycosylation-deficient, and truncated Pom152-GFP chimeras expressed in cells containing endogenous Pom152 localize to both NPCs and cortical endoplasmic reticulum (ER). Expression of Pom152-GFP fusions in pom152Δ cells results in detectable localization at only the NE by full-length and amino-terminal Pom152-GFP fusions, but continued retention at both the NE and ER for a chimera lacking just the carboxy-terminal 377 amino acids. Neither deletion of Pom152 nor its carboxy-terminal glycosylation sites altered the nuclear protein export rate of an Msn5/Kap142 protein cargo. These data narrow the Pom152 region sufficient for NPC localization and provide evidence that alterations in other domains may impact Pom152 targeting or affinity for the NPC.
Highlights
Nuclear pore complexes (NPCs) are large, aqueous, proteinaceous channels that perforate the nuclear envelope of eukaryotic cells and regulate communication and transport between the Biology Open Accepted manuscript nucleoplasm and cytoplasm (Beck and Hurt, 2017; Knockenhauer and Schwartz, 2016; Tran and Wente, 2006)
The amino-terminal and transmembrane domains together are sufficient for localization of Pom152 to the yeast NPC In order to further define the region of Pom152 sufficient for anchoring at the NPC, Pom152GFP chimeras containing either wild type POM152 (Pom152-GFP), the amino-terminal and TM sequences exclusively, a truncation of the carboxyterminal 377 amino acids, or POM152 lacking glycosylation sites at residues 280, 398, 569, and 1099 in the C-terminal lumenal domain (pom152Δglyc-GFP; (Belanger et al, 2005)) were transformed into wild type yeast (Fig. 1A)
These observations suggest that no portion of the lumenal carboxy-terminus of Pom152 is required for NPC localization, but that alterations in the carboxy-terminal domain may alter NPC affinity or targeting relative to wild type
Summary
Nuclear pore complexes (NPCs) are large, aqueous, proteinaceous channels that perforate the nuclear envelope of eukaryotic cells and regulate communication and transport between the Biology Open Accepted manuscript nucleoplasm and cytoplasm (Beck and Hurt, 2017; Knockenhauer and Schwartz, 2016; Tran and Wente, 2006). FG-Nups facilitate substrate translocation across the NPC via hydrophobic regions containing phenylalanine-glycine (FG) repeat sequences by forming a diffusion barrier within the pore that selectively allows passage of nuclear transport factors (NTFs) and their cargoes (Onischenko and Weis, 2011; Rout et al, 2000). Non-FG Nups form the structural scaffold with which FG-Nups associate within the NPC (Kim et al, 2018; Kosinski et al, 2016; Lin et al, 2016). The pore membrane Nups are Pom152, Pom, Pom, and Ndc, and each is involved in both assembly of nascent NPCs and maintenance of the structural organization of each NPC via association with each other and with non-FG Nups within the pore
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