Dynamics of nuclear pore distribution in nucleoporin mutant yeast cells.

To follow the dynamics of nuclear pore distribution in living yeast cells, we have generated fusion proteins between the green fluorescent protein (GFP) and the yeast nucleoporins Nup49p and Nup133p. In nup133- dividing cells that display a constitutive nuclear pore clustering, in vivo analysis of GFP-Nup49p localization revealed changes in the distribution of nuclear pore complex (NPC) clusters. Furthermore, upon induction of Nup133p expression in a GAL-nup133 strain, a progressive fragmentation of the NPC aggregates was observed that in turn led to a wild-type nuclear pore distribution. To try to uncouple Nup133p-induced NPC redistribution from successive nuclear divisions and nuclear pore biogenesis, we devised an assay based on the formation of heterokaryons between nup133- mutants and cells either expressing or overexpressing Nup133p. Under these conditions, the use of GFP-Nup133p and GFP-Nup49p fusion proteins revealed that Nup133p can be rapidly targeted to the clustered nuclear pores, where its amino-terminal domain is required to promote the redistribution of preexisting NPCs.

An evolutionarily conserved NPC subcomplex, which redistributes in part to kinetochores in mammalian cells.

The nuclear pore complexes (NPCs) are evolutionarily conserved assemblies that allow traffic between the cytoplasm and the nucleus. In this study, we have identified and characterized a novel human nuclear pore protein, hNup133, through its homology with the Saccharomyces cerevisiae nucleoporin scNup133. Two-hybrid screens and immunoprecipitation experiments revealed a direct and evolutionarily conserved interaction between Nup133 and Nup84/Nup107 and indicated that hNup133 and hNup107 are part of a NPC subcomplex that contains two other nucleoporins (the previously characterized hNup96 and a novel nucleoporin designated as hNup120) homologous to constituents of the scNup84 subcomplex. We further demonstrate that hNup133 and hNup107 are localized on both sides of the NPC to which they are stably associated at interphase, remain associated as part of a NPC subcomplex during mitosis, and are targeted at early stages to the reforming nuclear envelope. Throughout mitosis, a fraction of hNup133 and hNup107 localizes to the kinetochores, thus revealing an unexpected connection between structural NPCs constituents and kinetochores. Photobleaching experiments further showed that the mitotic cytoplasm contains kinetochore-binding competent hNup133 molecules and that in contrast to its stable association with the NPCs the interaction of this nucleoporin with kinetochores is dynamic.

Functional characterization of a Nup159p-containing nuclear pore subcomplex.

Nup159p/Rat7p is an essential FG repeat-containing nucleoporin localized at the cytoplasmic face of the nuclear pore complex (NPC) and involved in poly(A)+ RNA export and NPC distribution. A detailed structural-functional analysis of this nucleoporin previously demonstrated that Nup159p is anchored within the NPC through its essential carboxyl-terminal domain. In this study, we demonstrate that Nup159p specifically interacts through this domain with both Nsp1p and Nup82p. Further analysis of the interactions within the Nup159p/Nsp1p/Nup82p subcomplex using the nup82Delta108 mutant strain revealed that a deletion within the carboxyl-terminal domain of Nup82p prevents its interaction with Nsp1p but does not affect the interaction between Nup159p and Nsp1p. Moreover, immunofluorescence analysis demonstrated that Nup159p is delocalized from the NPC in nup82Delta108 cells grown at 37 degrees C, a temperature at which the Nup82Delta108p mutant protein becomes degraded. This suggests that Nup82p may act as a docking site for a core complex composed of the repeat-containing nucleoporins Nup159p and Nsp1p. In vivo transport assays further revealed that nup82Delta108 and nup159-1/rat7-1 mutant strains have little if any defect in nuclear protein import and protein export. Together our data suggest that the poly(A)+ RNA export defect previously observed in nup82 mutant cells might be due to the loss from the NPCs of the repeat-containing nucleoporin Nup159p.

Overexpression of yeast karyopherin Pse1p/Kap121p stimulates the mitochondrial import of hydrophobic proteins in vivo.

During evolution, cellular processes leading to the transfer of genetic information failed to send all the mitochondrial genes into the nuclear genome. Two mitochondrial genes are still exclusively located in the mitochondrial genome of all living organisms. They code for two highly hydrophobic proteins: the apocytochrome b and the subunit I of cytochrome oxidase. Assuming that the translocation machinery could not efficiently transport long hydrophobic fragments, we searched for multicopy suppressors of this physical blockage. We demonstrated that overexpression of Pse1p/Kap121p or Kap123p, which belong to the superfamily of karyopherin beta proteins, facilitates the translocation of chimeric proteins containing several stretches of apocytochrome b fused to a reporter mitochondrial gene. The effect of PSE1/KAP121 overexpression (in which PSE1 is protein secretion enhancer 1) on mitochondrial import of the chimera is correlated with an enrichment of the corresponding transcript in cytoplasmic ribosomes associated with mitochondria. PSE1/KAP121 overexpression also improves the import of the hydrophobic protein Atm1p, an ABC transporter of the mitochondrial inner membrane. These results suggest that in vivo PSE1/KAP121 overexpression facilitates, either directly or indirectly, the co-translational import of hydrophobic proteins into mitochondria.