Academic journal article Genetics

The Integral Membrane Protein Pom34p Functionally Links Nucleoporin Subcomplexes

Academic journal article Genetics

The Integral Membrane Protein Pom34p Functionally Links Nucleoporin Subcomplexes

Article excerpt

ABSTRACT

Here we have examined the function of Pom34p, a novel membrane protein in Saccharomyces cerevisiae, localized to nuclear pore complexes (NPCs). Membrane topology analysis revealed that Pom34p is a double-pass transmembrane protein with both the amino (N) and carboxy (C) termini positioned on the cytosolic/pore face. The network of genetic interactions between POM34 and genes encoding other nucleoporins was established and showed specific links between Pom34p function and Nup170p, Nup188p, Nup59p, Glc2p, Nup159p, and Nup82p. The transmembrane domains of Pom34p in addition to either the N- or C-terminal region were necessary for its function in different double mutants. We further characterized the pom34ΔN nup188Δ mutant and found it to be perturbed in both NPC structure and function. Mislocalization of a subset of nucleoporins harboring phenylalanine-glycine repeats was observed, and nuclear import capacity for the Kap104p and Kap121p pathways was inhibited. In contrast, the pom34Δ pom152Δ double mutant was viable at all temperatures and showed no such defects. Interestingly, POM152 overexpression suppressed the synthetic lethality of pom34Δ nup170Δ and pom34Δ nup59Δ mutants. We speculate that multiple integral membrane proteins, either within the nuclear pore domain or in the nuclear envelope, execute coordinated roles in NPC structure and function.

IN eukaryotic cells, translocation of macrornolecules across organellar membranes requires specialized protein complexes embedded at the sites of entry and/ or exit (SCHNELL and HEBERT 2003). Nucleocytoplasmic transport is mediated through nuclear pore complexes (NPCs) anchored in a nuclear envelope (NE) pore. These NPCs are formed from the assembly of at least 30 nucleoporins (Nups), in multiple copies each, that are organized in biochemically discrete subcomplexes and localized to specific NPC substructures (FAHRENKROG and AEBI 2003; SUNTHARALINGAM and WENTE 2003). In sum, NPCs have a predicted mass of ^44 MDa in Saccharomyces cerevisiae and 60 MDa in vertebrates (ROUT et al 2000; CRONSHAW et al 2002). Three-dimensional morphology studies have observed close contact between the proteinaceous NPC structure and surrounding NE (pore membrane), supporting models wherein nuclear pace jwembrane proteins (Poms) play key roles in organizing NPC architecture (ALLEN et al. 2000; STOFFLER et al. 2003; BECK et al. 2004). Moreover, the interactions between integral membrane proteins in the lumen are speculated to promote fusion of the inner and outer nuclear membranes for formation of the nuclear pore in the intact NE (GOLDBERG et al 1997). The mechanisms by which the membrane fusion and subsequent recruitment of distinct NPC subcomplexes occur have not been fully elucidated.

Two vertebrate Poms have been identified and characterized: gp210 and Poml21. Both are type I transmembrane proteins with single membrane-spanning segments and their amino (N)-terminal regions positioned in the NE lumen (WOZNIAK et al. 1989; GREBER et al. 1990; HALLBERG et al. 1993; SODERQVIST and HALLBERG 1994). However, they share no sequence homology and likely play distinct roles in NPC structure and function. Most of the gp210 mass is lumenally localized. In contrast, the majority of Poml21 is exposed to the cytosolic/pore side of the membrane. After integration into the endoplasmic reticulum (ER), gp210 and Poml21 are sorted to the nuclear pore membrane by lateral diffusion through the functionally continuous ER (YANG^ al. 1997; IMREH et al 2003). During postmitotic NE reformation in mammalian cells, Poml21 is recruited to the nuclear periphery at a very early stage, while the recruitment of gp210 occurs relatively late (BoDOOR et al. 1999). Fluorescence recovery after photobleaching experiments of interphase NPCs has shown that gp210 is notably dynamic with a relatively rapid exchange rate at NPCs. In contrast, Poml21 is markedly stable with a much slower exchange rate (ERIKSSON et al. …

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