No abstract

The establishment and maintenance of cellular polarity are essential biological processes that must be maintained throughout the lifetime of eukaryotic organisms. The Par-1 protein kinases are key polarity determinants that have been conserved throughout evolution. Par-1 directs anterior-posterior asymmetry in the one-cell C. elegans embryo and the Drosophila oocyte. In mammalian cells, Par-1 may regulate epithelial cell polarity. Relevant substrates of Par-1 in these pathways are just being identified, but it is not yet known how Par-1 itself is regulated. Here, we demonstrate that human Par-1b (hPar-1b) interacts with and is negatively regulated by atypical PKC. hPar-1b is phosphorylated by aPKC on threonine 595, a residue conserved in Par-1 orthologs in mammals, worms, and flies. The equivalent site in hPar-1a, T564, is phosphorylated in vivo and by aPKC in vitro. Importantly, phosphorylation of hPar-1b on T595 negatively regulates the kinase activity and plasma membrane localization of hPar-1b in vivo. This study establishes a novel functional link between two central determinants of cellular polarity, aPKC and Par-1, and suggests a model by which aPKC may regulate Par-1 in polarized cells.

Abstract. Nup116p is a member of a family of five yeast nuclear pore complex (NPC) proteins that share an amino terminal region of repetitive tetrapeptide "GLFG" motifs. Previous experiments characterized the unique morphological perturbations that occur in a nup116 null mutant: temperature-sensitive formation of nuclear envelope seals over the cytoplasmic face of the NPC (Wente, S. R., and G. Blobel. 1993. J. Cell Biol. 123:275-284). Three approaches have been taken to dissect the structural basis for Nupll6p's role in NPC function. First, deletion mutagenesis analysis of NUPll6 revealed that the GLFG region was required for NPC function. This was not true for the other four yeast GLFG family members (Nup49p, Nup57p, Nupl00p, and Nup145p). Moreover, deletion of either half of Nup116p's GLFG repeats or replacement of Nupll6p's GLFG region with either Nupl00p's GLFG region or Nsplp's FXFG repetitive region abolishes the function of Nupl16p. At a semipermissive growth temperature, the cells lacking Nupl16p's GLFG region displayed a diminished capacity for nuclear import. Second, overexpression of Nup116p's GLFG region severely inhibited cell growth, rapidly blocked polyadenylated-RNA export, and fragmented the nucleolus. Although it inhibited nuclear export, the overexpressed GLFG region appeared predominantly localized in the cytoplasm and NPC/nuclear envelope structure was not perturbed in thin section electron micrographs. Finally, using biochemical and two-hybrid analysis, an interaction was characterized between Nupl16p's GLFG region and Kap95p, an essential yeast homologue of the vertebrate nuclear import factor p97/Imp90/karopherin [3. These data show that Nup116p's GLFG region has an essential role in mediating nuclear transport.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin NuplOOp were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srplp, the nuclear localization signal receptor, as well as Riplp, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.

The mechanism of mRNA export is a complex issue central to cellular physiology. We characterized previously yeast Gle1p, a protein with a leucine-rich (LR) nuclear export sequence (NES) that is essential for poly(A) ؉ RNA export in Saccharomyces cerevisiae. To characterize elements of the vertebrate mRNA export pathway, we identified a human homologue of yeast Gle1p and analyzed its function in mammalian cells. hGLE1 encodes a predicted 75-kDa polypeptide with high sequence homology to yeast Gle1p, but hGle1p does not contain a sequence motif matching any of the previously characterized NESs. hGLE1 can complement a yeast gle1 temperature-sensitive export mutant only if a LR-NES is inserted into it. To determine whether hGle1p played a role in nuclear export, anti-hGle1p antibodies were microinjected into HeLa cells. In situ hybridization of injected cells showed that poly(A) ؉ RNA export was inhibited. In contrast, there was no effect on the nuclear import of a glucocorticoid receptor reporter. We conclude that hGle1p functions in poly(A) ؉ RNA export, and that human cells facilitate such export with a factor similar to yeast but without a recognizable LR-NES. With hGle1p localized at the nuclear pore complexes, hGle1p is positioned to act at a terminal step in the export of mature RNA messages to the cytoplasm.The nuclear export of proteins and ribonucleoprotein (RNP) particles through the nuclear pore complex (NPC) is a facilitated and signal-dependent process (1-3). Moreover, RNA processing and transport events are tightly coupled, such that splicing, polyadenylation, and capping all affect the export process (4-9). Throughout the processing and exit pathway, RNA is bound by distinct proteins and the critical signals for export are predicted to reside on these proteins (1, 2, 10). This has been demonstrated clearly in studies of the HIV-1 Rev protein, which specifically binds unspliced viral RNA (11-13). The RNA-binding domain of Rev is distinct from a region containing a leucine-rich (LR) nuclear export sequence (NES), which is both necessary and sufficient for mediating nuclear export (14, 15). The LR-NES is recognized in the nucleoplasm by a nuclear export receptor, Crm1p͞exportin (16), that is a member of a family of ␤ nuclear transport factors (17,18). Thus, the NES of Rev directs export of the protein and bound viral RNA coincidentally through the NPC by interaction with an exporting ␤.In vertebrate cells, different RNA classes are exported by independent pathways with each RNA type (mRNA, U snRNA, tRNA, or rRNA) potentially requiring at least a subset of distinct factors (reviewed in refs.