A small nuclear GTP-binding protein from tomato suppresses a Schizosaccharomyces pombe cell-cycle mutant.

Abstract

Ran is a 25-kDa Ras-related nuclear GTPbinding protein which is very highly conserved in humans, Saccharomyces cerevisiae, and Schizosaccharomyces pombe. Ran has been found to form a stable, noncovalent complex with the chromatin-associated protein RCC1, a negative regulator of mitosis. In Sch. pombe, a temperature-sensitive mutation in the RCC1 homolog encoded by thepiml gene causes premature induction of mitosis, and this mutation can be suppressed by overexpression of the Ran homolog encoded by spil. We report here the cloning of three Ran cDNAs from tomato. The Ras-related protein Ran is a 25-kDa nuclear protein that was shown in mammalian cells to bind to the chromatinassociated protein RCC1 (1). RCC1 was originally isolated from a hamster cell line (2) where a temperature-sensitive RCC1 mutation causes either premature chromosome condensation, activation ofp34cdc2 kinase, and entry into mitosis if the temperature is shifted during the S or G2 phase of the cell cycle or cell-cycle arrest if the temperature is shifted during G1 (3, 4). RCC1 catalyzes the exchange of guanine nucleotides on the Ran protein (5), suggesting that RCC1 and Ran may constitute parts of a GTPase switch that is involved in the coupling of the completion of DNA replication to the onset of M phase (for reviews, see refs. 6-8). Like their mammalian counterparts, the RCC1 and Ran homologs of Schizosaccharomyces pombe, called pimil and spil, respectively, have also been shown to be involved in the coupling of DNA replication to mitosis (9). A temperature-sensitive mutation in the piml gene causes premature chromosome condensation and entry into mitosis at the restrictive temperature. Overexpression of spil can suppress this mutation, and piml and spil have been shown to physically interact in vitro (10). In Saccharomyces cerevisiae a temperaturesensitive mutation in the RCC1 homolog PRP20/SRM1 can be suppressed by overexpression of either of the two S. cerevisiae Ran homologs (11,12). However, unlike RCC1 and piml, PRP20/SRM1 was first discovered as a gene essential for a variety of nuclear functions including pheromone response, mRNA metabolism and export, nuclear structure, and plasmid and chromosome stability (13-17). Also, H1 kinase is not activated in srml mutants (16), and disruption of the two Ran genes inhibits mRNA export (12), indicating that RCC1 and Ran play broad roles in nuclear structure and function in S. cerevisiae, which may differ from other organisms. However, findings that RCC1 is involved in mRNA export from mammalian nuclei (14) (22). Their sequences were 5'-GGGAATTCGGIGARTTYGAGAARAARTA-3' and 5'-CGGGATCCGGYTTYTCGAARTTRTARTT-3', where I is deoxyinosine. The PCR mixture contained 500 pmol of each primer, amplified phage library from tomato (1.2 x 107 plaque-forming units) or Arabidopsis (1.7 x 106 plaqueforming units), and 2.5 units of Taq DNA polymerase (Perkin-Elmer/Cetus). Forty-four cycles were performed of 1 min at 94°C, 2 min at the annealing temperature, and 2 min at 70°C. The annealing temperature star...

GAP Activity, but Not Subcellular Targeting, Is Required for Arabidopsis RanGAP Cellular and Developmental Functions

“…The 12 mutagenized RanGAP1 variants were tested for their ability to rescue the temperaturesensitive S. cerevisiae yeast strain rna1-1 (Corbett et al, 1995). As was shown previously, RanGAP1 restored growth of rna1-1 under restrictive conditions (37°C) (Ach and Gruissem, 1994;Joseph et al, 2002;Xu et al, 2007). Similarly, the RanGAP1 AAP variant, which has disrupted NE association and targeting to all mitotic sites (Rose and Meier, 2001;Zhao et al, 2008), rescued the growth phenotype under restrictive conditions.…”

“…It interacts with two nuclear envelope (NE)-associated coiled-coil protein families, the WIPs (WPP domain-interacting proteins) and the WITs (WPP-domain-interacting tail-anchored proteins) (Rose and Meier, 2001;Xu et al, 2007;Zhao et al, 2008), which are required for RanGAP NE localization. Arabidopsis thaliana RanGAP1 complements the temperaturesensitive Saccharomyces cerevisiae Rna1p mutant rna1-1, indicating that its GTPase activation (GAP) activity is conserved (Ach and Gruissem, 1994;Merkle et al, 1994;Pay et al, 2002). Arabidopsis contains two homologous copies of RanGAP, RanGAP1 and RanGAP2, which share 60% amino acid identity with each other and ;20% identity with either S. cerevisiae Rna1p or human RanGAP (Rose and Meier, 2001).…”

Direct and indirect association of the small GTPase ran with nuclear pore proteins and soluble transport factors: studies in Xenopus laevis egg extracts.

“…Ran is an extremely abundant protein (107 copies per HeLa cell; Bischoff and Ponstingl, 1991), whose amino acid sequence is well conserved across species (Ach and Gruissem, 1994). Ran is distinct from most other members of the Ras superfamily in at least two ways.…”

Identification and characterization of a lower plant Ypt/Rab guanosine dissociation inhibitor (GDI)

“…[36][37][38]). Nevertheless, the current progress encourages us to predict that, besides confirmation of established functions, fascinating novel aspects can evolve from the study of green organisms' Ypt/Rabs [39][40][41]. In accordance with this, we recently reported that Ypt proteins can be found not only in the cell body, but also in the flagella of the green algae V. carteri and C. reinhardtii [9], which is in line with a previous report already noting GTPase activities in Paramecium cilia [42].…”