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Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

van Es S, Wessels D, Soll DR, Borleis J, Devreotes PN - J. Cell Biol. (2001)

Bottom Line: Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype.TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker.The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

Show MeSH
Localization and function of truncated TorA–GFP proteins. Truncated TorA–GFP proteins were expressed in Ax3 to study localization of the fluorescent signal. The same constructs were expressed in torA− to study their ability to rescue this mutant.
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Figure 7: Localization and function of truncated TorA–GFP proteins. Truncated TorA–GFP proteins were expressed in Ax3 to study localization of the fluorescent signal. The same constructs were expressed in torA− to study their ability to rescue this mutant.

Mentions: To identify domains required for TorA function and localization, we made a series of COOH terminally truncated proteins, which were fused to GFP. To study whether these mutant proteins were functional, we tested whether expression of these constructs could suppress the torA− phenotype. As shown in Fig. 7, truncation of the 63 COOH-terminal amino acids results in the inability to restore the torA− phenotype, indicating that the protein is not functional. Interestingly, this protein and shorter NH2-terminal fragments that lack the coiled coil domain localize to mitochondrial clusters. As more of the COOH-terminal sequence was deleted, staining of the clusters decreases, and staining of the entire mitochondrion increases. The NH2-terminal 156 amino acids are necessary and sufficient to target GFP to the mitochondrion. When this region is deleted, the protein targets to the cytosol and does not complement the torA− mutant. Interestingly, the NH2 terminally truncated protein does not form clusters in the cytosol, suggesting that the formation of these structures requires the association with certain mitochondrial proteins.


Tortoise, a novel mitochondrial protein, is required for directional responses of Dictyostelium in chemotactic gradients.

van Es S, Wessels D, Soll DR, Borleis J, Devreotes PN - J. Cell Biol. (2001)

Localization and function of truncated TorA–GFP proteins. Truncated TorA–GFP proteins were expressed in Ax3 to study localization of the fluorescent signal. The same constructs were expressed in torA− to study their ability to rescue this mutant.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196008&req=5

Figure 7: Localization and function of truncated TorA–GFP proteins. Truncated TorA–GFP proteins were expressed in Ax3 to study localization of the fluorescent signal. The same constructs were expressed in torA− to study their ability to rescue this mutant.
Mentions: To identify domains required for TorA function and localization, we made a series of COOH terminally truncated proteins, which were fused to GFP. To study whether these mutant proteins were functional, we tested whether expression of these constructs could suppress the torA− phenotype. As shown in Fig. 7, truncation of the 63 COOH-terminal amino acids results in the inability to restore the torA− phenotype, indicating that the protein is not functional. Interestingly, this protein and shorter NH2-terminal fragments that lack the coiled coil domain localize to mitochondrial clusters. As more of the COOH-terminal sequence was deleted, staining of the clusters decreases, and staining of the entire mitochondrion increases. The NH2-terminal 156 amino acids are necessary and sufficient to target GFP to the mitochondrion. When this region is deleted, the protein targets to the cytosol and does not complement the torA− mutant. Interestingly, the NH2 terminally truncated protein does not form clusters in the cytosol, suggesting that the formation of these structures requires the association with certain mitochondrial proteins.

Bottom Line: Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype.TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker.The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.

ABSTRACT
We have identified a novel gene, Tortoise (TorA), that is required for the efficient chemotaxis of Dictyostelium discoideum cells. Cells lacking TorA sense chemoattractant gradients as indicated by the presence of periodic waves of cell shape changes and the localized translocation of cytosolic PH domains to the membrane. However, they are unable to migrate directionally up spatial gradients of cAMP. Cells lacking Mek1 display a similar phenotype. Overexpression of Mek1 in torA- partially restores chemotaxis, whereas overexpression of TorA in mek1- does not rescue the chemotactic phenotype. Regardless of the genetic background, TorA overexpressing cells stop growing when separated from a substrate. Surprisingly, TorA-green fluorescent protein (GFP) is clustered near one end of mitochondria. Deletion analysis of the TorA protein reveals distinct regions for chemotactic function, mitochondrial localization, and the formation of clusters. TorA is associated with a round structure within the mitochondrion that shows enhanced staining with the mitochondrial dye Mitotracker. Cells overexpressing TorA contain many more of these structures than do wild-type cells. These TorA-containing structures resist extraction with Triton X-100, which dissolves the mitochondria. The characterization of TorA demonstrates an unexpected link between mitochondrial function, the chemotactic response, and the capacity to grow in suspension.

Show MeSH