Novel G Proteins, Rag C and Rag D, Interact with GTP-binding Proteins, Rag A and Rag B

Abstract

Rag A/Gtr1p are G proteins and are known to be involved in the RCC1-Ran pathway. We employed the two-hybrid method using Rag A as the bait to identify proteins binding to Rag A, and we isolated two novel human G proteins, Rag C and Rag D. Rag C demonstrates homology with Rag D (81.1% identity) and with Gtr2p of Saccharomyces cerevisiae (46.1% identity), and it belongs to the Rag A subfamily of the Ras family. Rag C and Rag D contain conserved GTP-binding motifs (PM-1, -2, and -3) in their N-terminal regions. Recombinant glutathione S-transferase fusion protein of Rag C efficiently bound to both [ 3 H]GTP and [ 3 H]GDP. Rag A was associated with both Rag C and Rag D in their C-terminal regions where a potential leucine zipper motif and a coiled-coil structure were found. Rag C and D were associated with both the GDP and GTP forms of Rag A. Both Rag C and Rag D changed their subcellular localization, depending on the nucleotidebound state of Rag A. In a similar way, the disruption of S. cerevisiae GTR1 resulted in a change in the localization of Gtr2p.G proteins are a superfamily of regulatory GTP hydrolases and are composed of a large number of proteins. These include Ras family proteins, hetrotrimeric G protein ␣ subunits, and elongation factors TU and G, among others (1). Ras-like small G proteins such as Ras, Rab, Rho, ARF, and Ran are monomeric and bind to the guanine nucleotides, GTP or GDP, to function as molecular switches while also playing crucial roles in cell growth, differentiation, and protein traffic between different compartments within the cells (2, 3). Ras is a key regulator of cell growth and is an essential component of the signal transduction pathways initiated by receptor tyrosine kinase (4). The Rho family members consist of Rho, Rac, and Cdc42 subtypes that control the actin cytoskeleton and that play a role in the regulation of transcription (5). ADP-ribosylation factors play a role in the vesicular trafficking pathway (6). The Rab subfamily plays a role in secretory and endocytic pathways and is located within a distinct cellular compartment (7).Ran is a well characterized nuclear Ras-like small G protein that plays an essential role in the import and export of proteins and RNAs across the nuclear membrane through the nuclear pore complex (8) and also plays a role in the induction of microtubule self-organization in Xenopus egg extracts (9 -14). There are a large number of factors that interact with either the GDP-bound form or the GTP-bound form of Ran (Gsp1p), these being nucleoporin, RanBP2/NUP358 (15, 16), Prp20p interacting protein, RanBP3(Yrb2p) (17-19), the exosome involved in ribosomal RNA processing, Dis3p (20, 21), microtubule nucleation, RanBPM (22), and regulators of Ran, Ran-GAP1 (23-25), RanBP1(Yrb1p) (26, 27), RCC1/RanGEF (28), and Mog1p (29).RCC1 catalyzes guanine nucleotide exchange on Ran (30) and is located inside the nucleus, bound to chromatin (31). The concentration of GTP within the cell is ϳ30 times higher than the concentration of GDP, thus resulting in the prefer...

Crystal Structure of the Gtr1pGTP-Gtr2pGDP Protein Complex Reveals Large Structural Rearrangements Triggered by GTP-to-GDP Conversion

“…1C) as a result of a large conformational transition, which is described below in detail. This finding is consistent with a previous observation that Mg 2ϩ is not required for GDP binding to RagC (4). Biochemical studies suggested that the absence of Mg 2ϩ ion can increase the rate of GDP dissociation up to 1000-fold (11,12).…”

“…Rag GTPases exist as heterodimers containing a Gtr1p-like Rag GTPase (either RagA or RagB) and a Gtr2p-like Rag GTPase (either RagC or RagD) (4). The function of the heterodimeric complex relies on which guanine nucleotide is bound to each of the Rag GTPases.…”

“…The signal transduction through which amino acids activate TORC1 critically depends on the highly conserved Rag GTPases, which are members of the Ras GTPase superfamily (2,3). Mammalian cells express four members of Rag GTPases: RagA, RagB, RagC, and RagD (4). In yeast, Gtr1p and Gtr2p are the orthologues of RagA/B and RagC/D, respectively (5).…”

Cell growth control: little eukaryotes make big contributions

“…EGO1/MEH1, EGO3/NIR1/ SLM4, GTR1, or GTR2) or a decrease in TORC1 signalling , see also above) provides further circumstantial evidence for an intriguing model in which EGOC relays vacuolar nutrient signals to TORC1. Although the function of EGO1/MEH1 and EGO3/NIR1/SLM4 is not yet clear, GTR1 and GTR2 are small GTPases and the yeast orthologues of the mammalian RagA, -B, -C and -D proteins (Sekiguchi et al, 2001). Notably, RagC is upregulated in metastatic mammalian cells and suggested to be involved in malignant progression (Nakaji et al, 1999).…”

dRAGging Amino Acid-mTORC1 Signaling by SH3BP4

“…Rag GTPases are evolutionarily conserved throughout eukaryotes. Higher eukaryotes have four members of Rag GTPases: RagA, RagB, RagC and RagD (Sekiguchi et al, 2001). Rag GTPases exist in heterodimeric complexes consisting of RagA or RagB and RagC or RagD (Dubouloz et al, 2005;Hirose et al, 1998;Kim et al, 2008).…”