Phenotype of the fission yeast cell cycle regulatory mutant pim1‐46 is suppressed by a tobacco cDNA encoding a small, Ran‐like GTP‐binding protein

Abstract

Mutations in which the onset of mitosis is uncoupled from the completion of DNA replication has recently been described. Characterization of these mutants led to the identification of Pim1/Spi1 in fission yeast and RCC1/Ran proteins in mammalian cells. Their Saccharomyces cerevisae homologues, the MTR1/CNR1 proteins, appear to be involved in controlling RNA metabolism and transport. Here the isolation and partial characterization of plant cDNA clones which encode proteins homologous to the mammalian/fission yeast/budding yeast Ran/Spi/CNR proteins are reported. Higher plants appear to contain more than one gene per haploid genome which codes for Ran proteins. These genes are expressed in different plant tissues, including root tips and stems, known to contain mitotically active cells. The tobacco Ran-like proteins, like their mammalian and yeast homologues, are soluble proteins which are found in the cytoplasm and in the nucleus. In addition, it has been shown that overexpression of the tobacco Nt-Ran-A1 cDNA suppressed the phenotype of the temperature-sensitive fission yeast pim1-46 mutant. These results suggest that the plant Ran genes can be functionally equivalent to the mammalian/fission yeast/budding yeast Ran/Spi/CNR genes and that they may play a role: (i) in maintaining a coordinated cell cycle; (ii) in controlling RNA metabolism and transport in higher plants; and/or (iii) in protein import into the nucleus.

Protein Targeting to the Nuclear Pore. What Can We Learn from Plants?1

“…Permeabilization allows most cytosolic proteins to leak out of the cell, and it allows large molecules, such as antibodies, to enter the cell. In this assay, antibodies to proteins that are supposed to go to the nucleus are added to permeabilized protoplasts, and import of the antigen-antibody complex is measured by protease protection assays of the intranuclear antibody by immunoblot analysis (Harter et al, 1994). The ␣-subunit binds to NLSs and the ␤-subunit mediates NPC docking.…”

“…Ran homologs that are approximately 75% identical to other Ran homologs found in vertebrates and fungi have been identified in Arabidopsis (Haizel et al, 1997), tomato (Ach and Gruissem, 1994), tobacco (Merkle et al, 1994), fava bean (Saalbach and Christov, 1994), and Lotus japonicus (Borg et al, 1997). In plants Ran appears to be encoded by a small gene family whose members are expressed ubiquitously in various organs (Ach and Gruissem, 1994;Merkle et al, 1994;Haizel et al, 1997). As in mammals and yeast, Ran localizes to the nucleus in plant cells (Ach and Gruissem, 1994;Merkle et al, 1994).…”

“…In plants Ran appears to be encoded by a small gene family whose members are expressed ubiquitously in various organs (Ach and Gruissem, 1994;Merkle et al, 1994;Haizel et al, 1997). As in mammals and yeast, Ran localizes to the nucleus in plant cells (Ach and Gruissem, 1994;Merkle et al, 1994). A mutation in the Schizosaccharomyces pombe Ran gene pim1 has a cell-cycle defect that causes chromosomes to condense during DNA replication.…”

“…A mutation in the Schizosaccharomyces pombe Ran gene pim1 has a cell-cycle defect that causes chromosomes to condense during DNA replication. Overexpression of Ran cDNAs from tomato or tobacco suppresses the pim1 phenotype, indicating that these Ran proteins have a function in plants similar to that in yeast (Ach and Gruissem, 1994;Merkle et al, 1994). However, a direct role of plant Ran-like proteins in nuclear import and export is yet to be described.…”

Mechanisms of plant spindle formation

“…Ran itself is highly conserved in plants and other species (Ach and Gruissem 1994;Merkle et al 1994;Haizel et al 1997). As in animals, green fluorescence protein (GFP)-tagged Ran localizes to nuclei in tobacco cells (Yano et al 2006).…”