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Kap120 functions as a nuclear import receptor for ribosome assembly factor Rpf1 in yeast.

ABSTRACT: The nucleocytoplasmic exchange of macromolecules is mediated by receptors specialized in passage through the nuclear pore complex. The majority of these receptors belong to the importin beta protein family, which has 14 members in Saccharomyces cerevisiae. Nine importins carry various cargos from the cytoplasm into the nucleus, whereas four exportins mediate nuclear export. Kap120 is the only receptor whose transport cargo has not been found previously. Here, we characterize Kap120 as an importin for the ribosome maturation factor Rpf1, which was identified in a two-hybrid screen. Kap120 binds directly to Rpf1 in vitro and is released by Ran-GTP. At least three parallel import pathways exist for Rpf1, since nuclear import is defective in strains with the importins Kap120, Kap114, and Nmd5 deleted. Both kap120 and rpf1 mutants accumulate large ribosomal subunits in the nucleus. The nuclear accumulation of 60S ribosomal subunits in kap120 mutants is abolished upon RPF1 overexpression, indicating that Kap120 does not function in the actual ribosomal export step but rather in import of ribosome maturation factors.

SUBMITTER: Caesar S

PROVIDER: S-EPMC1446960 | biostudies-literature |

REPOSITORIES: biostudies-literature

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A RanGTP-independent mechanism allows ribosomal protein nuclear import for ribosome assembly.

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Transportin-1: A Nuclear Import Receptor with Moonlighting Functions.

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The yeast protein Mam33 functions in the assembly of the mitochondrial ribosome.

Project description:Mitochondrial ribosomes are functionally specialized for the synthesis of several essential inner membrane proteins of the respiratory chain. Although remarkable progress has been made toward understanding the structure of mitoribosomes, the pathways and factors that facilitate their biogenesis remain largely unknown. The long unstructured domains of unassembled ribosomal proteins are highly prone to misfolding and often require dedicated chaperones to prevent aggregation. To date, chaperones that ensure safe delivery to the assembling ribosome have not been identified in the mitochondrion. In this study, a respiratory synthetic lethality screen revealed a role for an evolutionarily conserved mitochondrial matrix protein called Mam33 in Saccharomyces cerevisiae mitoribosome biogenesis. We found that the absence of Mam33 results in misassembled, aggregated ribosomes and a respiratory lethal phenotype in combination with other ribosome-assembly mutants. Using sucrose gradient sedimentation, native affinity purifications, in vitro binding assays, and SILAC-based quantitative proteomics, we found that Mam33 does not associate with the mature mitoribosome, but directly binds a subset of unassembled large subunit proteins. Based on these data, we propose that Mam33 binds specific mitoribosomal proteins to ensure proper assembly.

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Project description:Here we profiled small RNAs from whole cell, cytoplasmic and nuclear extracts from three-week-old Arabidopsis seedlings. We unexpectedly found that nuclear functional hc-siRNAs are predominantly present in the cytoplasm. Samples from Arabidopsis thaliana whole cell, cytoplasmic and nuclear extracts with 3 replicates for each. 9 samples in all.

2012-07-12 | E-GEOD-36500 | biostudies-arrayexpress

Transportin2 functions as importin and mediates nuclear import of HuR.

Project description:The RanGTP-binding nuclear transport receptors transportin1 (TRN1) and transportin2 (TRN2) are highly similar in sequence but are reported to function in nuclear import and export, respectively. Here we show that TRN2 possesses properties of a nuclear import receptor. TRN1/2 both interacted with a similar set of RNA-binding proteins in a RanGTP-sensitive manner. TRN2 bound RanGTP with high affinity, a feature of nuclear import receptors. As expected of an import complex, RanGTP also disrupted the interaction between TRN2 and HuR, an RNA-binding protein previously described as a TRN2 export substrate. The HuR nucleocytoplasmic shuttling signal, a sequence resembling the M9 nuclear import signal of hnRNP A1, was necessary and sufficient for TRN-mediated nuclear import of HuR in vitro. Finally, crosscompetition experiments demonstrated that HuR nucleocytoplasmic shuttling signal and M9 are imported along redundant pathways involving TRN1/2, substantiating the function of TRN2 in nuclear import.

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Cytoplasmic Assembly and Selective Nuclear Import of Arabidopsis ARGONAUTE4/siRNA Complexes

2012-07-12 | GSE36500 | GEO

The ribosome assembly factor Nop53 controls association of the RNA exosome with pre-60S particles in yeast.

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Project description:The mitochondrial import receptor Tom70 (translocase of the mitochondrial outer membrane 70) interacts with chaperone-preprotein complexes through two domains: one that binds Hsp70 (heat-shock protein 70)/Hsc70 (heat-shock cognate 70) and Hsp90, and a second that binds preproteins. The oligomeric state of Tom70 has been controversial, with evidence for both monomeric and homodimeric forms. In the present paper, we report that the functional state of human Tom70 appears to be a monomer with mechanistic implications for its function in mitochondrial protein import. Based on analytical ultracentrifugation, cross-linking, size-exclusion chromatography and multi-angle light scattering, we found that the soluble cytosolic fragment of human Tom70 exists in equilibrium between monomer and dimer. A point mutation introduced at the predicted dimer interface increased the percentage of monomeric Tom70. Although chaperone docking to the mutant was the same as to the wild-type, the mutant was significantly more active in preprotein targeting. Cross-linking also demonstrated that the mutant formed stronger contacts with preprotein. However, cross-linking of full-length wild-type Tom70 on the mitochondrial membrane showed little evidence of homodimers. These results indicate that the Tom70 monomers are the functional form of the receptor, whereas the homodimers appear to be a minor population, and may represent an inactive state.

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Functional domains in nuclear import factor p97 for binding the nuclear localization sequence receptor and the nuclear pore.

Project description:The interaction of the nuclear protein import factor p97 with the nuclear localization sequence (NLS) receptor, the nuclear pore complex, and Ran/TC4 is important for coordinating the events of protein import to the nucleus. We have mapped the binding domains on p97 for the NLS receptor and the nuclear pore. The NLS receptor-binding domain of p97 maps to the C-terminal 60% of the protein between residues 356 and 876. The pore complex-binding domain of p97 maps to residues 152-352. The pore complex-binding domain overlaps the Ran-GTP- and Ran-GDP-binding domains on p97, but only Ran-GTP competes for docking in permeabilized cells. The N-ethylmaleimide sensitivity of the p97 for docking was investigated and found to be due to inhibition of p97 binding to the pore complex and to the NLS receptor. Site-directed mutagenesis of conserved cysteine residues in the pore- and receptor-binding domains identified two cysteines, C223 and C228, that were required for p97 to bind the nuclear pore. Inhibition studies on docking and accumulation of a NLS protein provided additional evidence that the domains identified biochemically are the functional domains involved in protein import. Together, these results suggest that Ran-GTP dissociates the receptor complex and prevents p97 binding to the pore by inducing a conformational change in the structure of p97 rather than simple competition for binding sites.

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Transportin-SR, a nuclear import receptor for SR proteins.

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| S-EPMC2133142 | biostudies-literature

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