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Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Davidson R, Laporte D, Wu JQ - Mol. Biol. Cell (2014)

Bottom Line: Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells.Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

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Rgf3 physically interacts with Art1 and is important for Art1 localization to the division site. (A–C) Overexpression of Rho1 or Rgf3 rescues the lysis phenotype of art1∆. (A) art1∆ cells with empty vector (JW3632) or pRho1 plasmid (JW3633). (B) art1∆ (JW2543) and 3nmt1-rgf3 art1∆ (JW3868) cells were grown in YE5S liquid medium for 40 h. (C) Quantification of lysis in cells as shown in A and B. (D and E) Art1 and Rgf3 co‑IP independent of the phosphorylation status of proteins. (D) Rgf3 (top; strain JW2421) and Art1 (bottom; strain JW3381) are phosphoproteins. (E) Rgf3 co‑IPs with Art1. IPs using anti-YFP antibody and cell lysates of strains expressing Art1-2YFP (JW3711), Rgf3-13Myc (JW2421), and Art1‑2YFP Rgf3-13Myc (JW2696) with or without λ-phosphatase. (F) Art1 and Rgf3 interact in the yeast two-hybrid assays. X-gal overlay assay between VP16-activation domain (AD)-Rgf3 and GAL4-DNA-binding domain (BD)-Art1 along with empty vector controls. The dark blue color indicates a positive reaction. (G and H) Rgf3 is important for Art1 localization. art1-mECitrine sad1-mCFP (JW2694) and art1-mECitrine sad1-mCFP N-degron-rgf3 strains (JW3351) were grown at 25°C and shifted to 36°C for 4 h before imaging at 36°C. (G) Art1 localizes to the contractile ring (arrowheads) and septal region (arrows) in wt cells. Asterisks indicate cells with a complete septum that lack Art1 signal at the division site. (H) Quantification of dividing cells (cells with two separated SPBs) with Art1-mECitrine signal at the division site. Scale bars: 5 μm.
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Figure 3: Rgf3 physically interacts with Art1 and is important for Art1 localization to the division site. (A–C) Overexpression of Rho1 or Rgf3 rescues the lysis phenotype of art1∆. (A) art1∆ cells with empty vector (JW3632) or pRho1 plasmid (JW3633). (B) art1∆ (JW2543) and 3nmt1-rgf3 art1∆ (JW3868) cells were grown in YE5S liquid medium for 40 h. (C) Quantification of lysis in cells as shown in A and B. (D and E) Art1 and Rgf3 co‑IP independent of the phosphorylation status of proteins. (D) Rgf3 (top; strain JW2421) and Art1 (bottom; strain JW3381) are phosphoproteins. (E) Rgf3 co‑IPs with Art1. IPs using anti-YFP antibody and cell lysates of strains expressing Art1-2YFP (JW3711), Rgf3-13Myc (JW2421), and Art1‑2YFP Rgf3-13Myc (JW2696) with or without λ-phosphatase. (F) Art1 and Rgf3 interact in the yeast two-hybrid assays. X-gal overlay assay between VP16-activation domain (AD)-Rgf3 and GAL4-DNA-binding domain (BD)-Art1 along with empty vector controls. The dark blue color indicates a positive reaction. (G and H) Rgf3 is important for Art1 localization. art1-mECitrine sad1-mCFP (JW2694) and art1-mECitrine sad1-mCFP N-degron-rgf3 strains (JW3351) were grown at 25°C and shifted to 36°C for 4 h before imaging at 36°C. (G) Art1 localizes to the contractile ring (arrowheads) and septal region (arrows) in wt cells. Asterisks indicate cells with a complete septum that lack Art1 signal at the division site. (H) Quantification of dividing cells (cells with two separated SPBs) with Art1-mECitrine signal at the division site. Scale bars: 5 μm.

Mentions: Our previous study showed that the art1-s34 mutant can be rescued by overexpression of Rho-GEFs Rgf1, Rgf2, and Rgf3 or Rho1 GTPase (Wu et al., 2010), but the mechanism is unknown. Rgf1 and Rgf2 have localizations and/or deletion phenotypes different from those of Art1 (Figures 1 and 2; Mutoh et al., 2005; Garcia et al., 2006; Wu et al., 2010). Thus we focused on Rgf3. Rgf3 is a known GEF for Rho1 that activates the cell wall–synthesizing enzymes β-glucan synthases and other effectors (Arellano et al., 1997, 1999; Nakano et al., 1997; Tajadura et al., 2004; Mutoh et al., 2005). We hypothesized that Art1 functions in the same pathway as Rgf3 and Rho1 for cell wall synthesis and cell integrity. To test this hypothesis, we investigated whether art1∆ cells can be rescued by Rgf3 or Rho1 overexpression. Cell lysis of art1∆ cells decreased to ≤ 5% when Rho1 or Rgf3 was overexpressed (Figure 3, A–C). In addition, we found that art1∆ was synthetic lethal at 25°C with rgf3-s44, a point mutation in the GEF domain of Rgf3 (Wu et al., 2010). Thus Art1 indeed functions in the same or a related pathway as Rgf3 and Rho1. Rho1 localizes to both the division site and cell tips (Arellano et al., 1997; Mutoh et al., 2005), whereas Art1 and Rgf3 only concentrate at the division site (Figure 2); thus it is more likely that Art1 interacts with Rgf3.


Regulation of Rho-GEF Rgf3 by the arrestin Art1 in fission yeast cytokinesis.

Davidson R, Laporte D, Wu JQ - Mol. Biol. Cell (2014)

Rgf3 physically interacts with Art1 and is important for Art1 localization to the division site. (A–C) Overexpression of Rho1 or Rgf3 rescues the lysis phenotype of art1∆. (A) art1∆ cells with empty vector (JW3632) or pRho1 plasmid (JW3633). (B) art1∆ (JW2543) and 3nmt1-rgf3 art1∆ (JW3868) cells were grown in YE5S liquid medium for 40 h. (C) Quantification of lysis in cells as shown in A and B. (D and E) Art1 and Rgf3 co‑IP independent of the phosphorylation status of proteins. (D) Rgf3 (top; strain JW2421) and Art1 (bottom; strain JW3381) are phosphoproteins. (E) Rgf3 co‑IPs with Art1. IPs using anti-YFP antibody and cell lysates of strains expressing Art1-2YFP (JW3711), Rgf3-13Myc (JW2421), and Art1‑2YFP Rgf3-13Myc (JW2696) with or without λ-phosphatase. (F) Art1 and Rgf3 interact in the yeast two-hybrid assays. X-gal overlay assay between VP16-activation domain (AD)-Rgf3 and GAL4-DNA-binding domain (BD)-Art1 along with empty vector controls. The dark blue color indicates a positive reaction. (G and H) Rgf3 is important for Art1 localization. art1-mECitrine sad1-mCFP (JW2694) and art1-mECitrine sad1-mCFP N-degron-rgf3 strains (JW3351) were grown at 25°C and shifted to 36°C for 4 h before imaging at 36°C. (G) Art1 localizes to the contractile ring (arrowheads) and septal region (arrows) in wt cells. Asterisks indicate cells with a complete septum that lack Art1 signal at the division site. (H) Quantification of dividing cells (cells with two separated SPBs) with Art1-mECitrine signal at the division site. Scale bars: 5 μm.
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Figure 3: Rgf3 physically interacts with Art1 and is important for Art1 localization to the division site. (A–C) Overexpression of Rho1 or Rgf3 rescues the lysis phenotype of art1∆. (A) art1∆ cells with empty vector (JW3632) or pRho1 plasmid (JW3633). (B) art1∆ (JW2543) and 3nmt1-rgf3 art1∆ (JW3868) cells were grown in YE5S liquid medium for 40 h. (C) Quantification of lysis in cells as shown in A and B. (D and E) Art1 and Rgf3 co‑IP independent of the phosphorylation status of proteins. (D) Rgf3 (top; strain JW2421) and Art1 (bottom; strain JW3381) are phosphoproteins. (E) Rgf3 co‑IPs with Art1. IPs using anti-YFP antibody and cell lysates of strains expressing Art1-2YFP (JW3711), Rgf3-13Myc (JW2421), and Art1‑2YFP Rgf3-13Myc (JW2696) with or without λ-phosphatase. (F) Art1 and Rgf3 interact in the yeast two-hybrid assays. X-gal overlay assay between VP16-activation domain (AD)-Rgf3 and GAL4-DNA-binding domain (BD)-Art1 along with empty vector controls. The dark blue color indicates a positive reaction. (G and H) Rgf3 is important for Art1 localization. art1-mECitrine sad1-mCFP (JW2694) and art1-mECitrine sad1-mCFP N-degron-rgf3 strains (JW3351) were grown at 25°C and shifted to 36°C for 4 h before imaging at 36°C. (G) Art1 localizes to the contractile ring (arrowheads) and septal region (arrows) in wt cells. Asterisks indicate cells with a complete septum that lack Art1 signal at the division site. (H) Quantification of dividing cells (cells with two separated SPBs) with Art1-mECitrine signal at the division site. Scale bars: 5 μm.
Mentions: Our previous study showed that the art1-s34 mutant can be rescued by overexpression of Rho-GEFs Rgf1, Rgf2, and Rgf3 or Rho1 GTPase (Wu et al., 2010), but the mechanism is unknown. Rgf1 and Rgf2 have localizations and/or deletion phenotypes different from those of Art1 (Figures 1 and 2; Mutoh et al., 2005; Garcia et al., 2006; Wu et al., 2010). Thus we focused on Rgf3. Rgf3 is a known GEF for Rho1 that activates the cell wall–synthesizing enzymes β-glucan synthases and other effectors (Arellano et al., 1997, 1999; Nakano et al., 1997; Tajadura et al., 2004; Mutoh et al., 2005). We hypothesized that Art1 functions in the same pathway as Rgf3 and Rho1 for cell wall synthesis and cell integrity. To test this hypothesis, we investigated whether art1∆ cells can be rescued by Rgf3 or Rho1 overexpression. Cell lysis of art1∆ cells decreased to ≤ 5% when Rho1 or Rgf3 was overexpressed (Figure 3, A–C). In addition, we found that art1∆ was synthetic lethal at 25°C with rgf3-s44, a point mutation in the GEF domain of Rgf3 (Wu et al., 2010). Thus Art1 indeed functions in the same or a related pathway as Rgf3 and Rho1. Rho1 localizes to both the division site and cell tips (Arellano et al., 1997; Mutoh et al., 2005), whereas Art1 and Rgf3 only concentrate at the division site (Figure 2); thus it is more likely that Art1 interacts with Rgf3.

Bottom Line: Using an Rgf3 conditional mutant and mislocalization experiments, we found that Art1 and Rgf3 are interdependent for localization to the division site.As expected, active Rho1 levels at the division site are reduced in art1∆ and rgf3 mutant cells.Taken together, these data reveal that the arrestin family protein Art1 regulates the protein levels and localization of the Rho-GEF Rgf3, which in turn modulates active Rho1 levels during fission yeast cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program of Molecular, Cellular, and Developmental Biology, The Ohio State University, Columbus, OH 43210 Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210.

Show MeSH
Related in: MedlinePlus