Academic journal article Genetics

Association of the GTP-Binding Protein Gtr1p with Rpc19p, a Shared Subunit of RNA Polymerase I and III in Yeast Saccharomyces Cerevisiae

Academic journal article Genetics

Association of the GTP-Binding Protein Gtr1p with Rpc19p, a Shared Subunit of RNA Polymerase I and III in Yeast Saccharomyces Cerevisiae

Article excerpt

ABSTRACT

Yeast Gtr1p and its human homolog RRAG A belong to the Ras-like small G-protein superfamily and genetically interact with RCC1, a guanine nucleotide exchange factor for Ran GTPase. Little is known regarding the function of Gtr1p. We performed yeast two-hybrid screening using Gtr1p as the bait to find interacting proteins. Rpc19p, a shared subunit of RNA polymerases I and III, associated with Gtr1p. The association of Gtr1p with Rpc19p occurred in a GTP-form-specific manner. RRAG A associated with RPA16 (human Rpc19p homolog) in a GTP-form-specific manner, suggesting that the association is conserved during evolution. Ribosomal RNA and tRNA synthesis were reduced in the gtr1Δ strain expressing the GDP form of Gtr1p, but not the GTP form of Gtr1p. Gel-filtration studies revealed an accumulation of the smaller Rpc19p-containing complex, but not of A135, in the gtr1Δ strain. Here, we propose that Gtr1p is involved in RNA polymerase I and III assembly by its association with Rpc19p and could be a mediator that links growth regulatory signals with ribosome biogenesis.

GUANINE nucleotide-binding proteins are a superfamily of regulatory GTP hydrolases composed of a large number of proteins, which include Ras family proteins, heterotrimeric G-protein á-subunits, and elongation factors TU and G, among others (SPRANG 1997). They have crucial roles in cell growth, proliferation, differentiation, and macromolecular trafficking across different intracellular compartments (MiLBURN et al. 1990; EXTON 1998). In yeast, Ras-like small G-proteins, including Raslp, Yptlp, Cdc42p, Arflp, Gtrlp, and Gsplp family proteins, bind to the guanine nucleotides GTP and GDP to function as molecular switches. Heterodimer formation of Gtrlp with Gtr2p is a feature that differs from other small G-proteins, which are monomeric.

Among the Ras superfamily of small G-proteins, Ran/ Gsplp is a nuclear protein with several functions, including nucleocytoplasmic transport of many types of protein and nucleic acids (NiSHiMOTO 2000; SAZER and DASSO 2000). The guanine nucleotide exchange factor for Ran/Gsplp (RCCl/Prp20p) is confined within the nucleus ( QUIMBY et al. 2000; NEMERGUT et al. 2001; Li et al. 2003), whereas the Ran GTPase-activating protein (RanGAP/Rnalp) is located in the cytosol. Compartmentalization of these factors is believed to create a gradient of GTPase Ran across the nuclear pore complex, which controls the stability of importin-â interactions with particular cargo molecules. The Ran gradient is also a key factor that controls mitotic processes, including spindle assembly during metaphase and reformation of the nuclear envelope during telophase (AZUMA and DASSO 2000; HEALD and WEIS 2000; QUIMBY et al 2000).

A cold-sensitive mutant of GTRl, gtrl-11, was identified as a suppressor of mtrl-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCCl homolog and rnal-1, a temperature-sensitive mutant of Gsplp GTPase-activating protein (NAKASHIMA et al. 1996). Gtrlp genetically interacts with Pho84p, a phosphate transporter (BuN et al. 1992). Gtrlp forms complexes with itself and Gtr2p, a member of the Gtrlp subfamily of Ras-like small G-proteins, and negatively regulates the Ran/Gsplp cycle through Gtr2p (NAKASHIMA et al. 1999). RRAG A/Rag A is a functional human homolog of Gtrlp (HiROSE et al 1998) that interacts with RRAG C/Rag C and RRAG D/Rag D GTP-binding proteins (SEKIGUCHI et al 2001), as well as NOP132 nucleolar protein (SEKIGUCHI et al. 2004). The yeast NopSp is a Nip7p-interacting protein involved in 60S ribosome biogenesis that also interacts with Gtrlp (Éôï et al. 2000; SEKIGUCHI et al 2004). In NopSp-depleted cells, pre-ribosomal RNA (rRNA) processing is abnormal (ZANCHIN and GOLDFARE 1999). S. cerevisiae Nip7p is required for efficient 60S ribosome biogenesis and is conserved evolutionarily (ZANCHIN et al 1997).

In the yeast S. cerevisiae, as in other eukaryotes, synthesis of rRNA transcripts accounts for 60% of the total transcriptional activity of rapidly growing yeast cells, which takes place in the nucleolus and is catalyzed by RNA polymerase I (pol I), which contains 14 distinct polypeptides (BUHLER et al. …

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