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

Pom33 is an integral membrane protein of the yeast nuclear pore complex (NPC), and it is required for proper NPC distribution and assembly. To characterize the Pom33 NPC-targeting determinants, we performed immunoprecipitation experiments followed by mass spectrometry analyses. This identified a new Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between the 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). Although deletion of Pom33 C-terminal domain (Pom33(ΔCTD)-GFP) impaired Pom33 stability and NPC targeting, mutants affecting either Kap123 binding or the amphipathic properties of the α-helices did not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects targeting of Pom33 to NPCs. These data highlight the requirement of multiple determinants and mechanisms for proper NPC localization of Pom33.

Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae.

Eukaryotic release factor 3 (eRF3) is implicated in translation termination and also interacts with the poly(A)-binding protein (PABP, Pab1 in yeast), a major player in mRNA metabolism. Despite conservation of this interaction, its precise function remains elusive. First, we showed experimentally that yeast eRF3 does not contain any obvious consensus PAM2 (PABP-interacting motif 2). Thus, in yeast this association is different from the well described interaction between the metazoan factors. To gain insight into the exact function of this interaction, we then analyzed the phenotypes resulting from deleting the respective binding domains. Deletion of the Pab1 interaction domain on eRF3 did not affect general mRNA stability or nonsense-mediated mRNA decay (NMD) pathway and induced a decrease in translational readthrough. Furthermore, combined deletions of the respective interacting domains on eRF3 and on Pab1 were viable, did not affect Pab1 function in mRNA stability and harbored an antisuppression phenotype. Our results show that in Saccharomyces cerevisiae the role of the Pab1 C-terminal domain in mRNA stability is independent of eRF3 and the association of these two factors negatively regulates translation termination.

Fifty years of nuclear pores and nucleocytoplasmic transport studies: multiple tools revealing complex rules.

Nuclear pore complexes (NPCs) are multiprotein assemblies embedded within the nuclear envelope and involved in the control of the bidirectional transport of proteins and ribonucleoparticles between the nucleus and the cytoplasm. Since their discovery more than 50 years ago, NPCs and nucleocytoplasmic transport have been the focus of intense research. Here, we review how the use of a multiplicity of structural, biochemical, genetic, and cell biology approaches have permitted the deciphering of the main features of this macromolecular complex, its mode of assembly as well as the rules governing nucleocytoplasmic exchanges. We first present the current knowledge of the ultrastructure of NPCs, which reveals that they are modular and repetitive assemblies of subunits referred to as nucleoporins, associated into stable subcomplexes and composed of a limited set of protein domains, including phenylalanine-glycine (FG) repeats and membrane-interacting domains. The outcome of investigations on nucleocytoplasmic trafficking will then be detailed, showing how it involves a limited number of molecular factors and common mechanisms, namely (i) indirect association of cargos with nuclear pores through receptors in the donor compartment, (ii) progression within the channel through dynamic hydrophobic interactions with FG-Nups, and (iii) NTPase-driven remodeling of transport complexes in the target compartment. Finally, we also discuss the outcome of more recent studies, which indicate that NPCs and the transport machinery are dynamic and versatile devices, whose biogenesis is tightly coordinated with the cell cycle, and which carry nonconventional duties, in particular, in mitosis, gene expression, and genetic stability.