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The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi.

Gillingham AK, Tong AH, Boone C, Munro S - J. Cell Biol. (2004)

Bottom Line: Bornens. 2004.Cell. 118:323-335).In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK.

ABSTRACT
Rud3p is a coiled-coil protein of the yeast cis-Golgi. We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal "GRIP-related Arf-binding" (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin (Rios, R.M, A. Sanchis, A.M. Tassin, C. Fedriani, and M. Bornens. 2004. Cell. 118:323-335). In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

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The COOH-terminal region of human GMAP-210 mediates targeting to the Golgi rather than centrosomes. (A) Confocal micrographs of COS cells expressing the NH2-terminal 372 amino acids of GMAP-210 COOH-terminally tagged with GFP, or the COOH-terminal 223 amino acids of GMAP-210 NH2-terminally tagged with GFP, or full-length GMAP-210 with an NH2-terminal myc tag. Cells were also labeled with antibodies against the endogenous Golgi protein GM130 or γ-tubulin. At very high levels of GMAP-210, normal Golgi morphology (arrow) is lost and the Golgi becomes fragmented as described previously (Pernet-Gallay et al., 2002), and yet γ-tubulin is still only on the centrosome (arrowheads indicate the centrosomes shown in merged inset). (B and C) Illustrative confocal images of COS cells expressing portions of GMAP-210 fused to GFP and costained with GM130 as in A, along with a summary of the results from all GMAP-210 portions examined.
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fig7: The COOH-terminal region of human GMAP-210 mediates targeting to the Golgi rather than centrosomes. (A) Confocal micrographs of COS cells expressing the NH2-terminal 372 amino acids of GMAP-210 COOH-terminally tagged with GFP, or the COOH-terminal 223 amino acids of GMAP-210 NH2-terminally tagged with GFP, or full-length GMAP-210 with an NH2-terminal myc tag. Cells were also labeled with antibodies against the endogenous Golgi protein GM130 or γ-tubulin. At very high levels of GMAP-210, normal Golgi morphology (arrow) is lost and the Golgi becomes fragmented as described previously (Pernet-Gallay et al., 2002), and yet γ-tubulin is still only on the centrosome (arrowheads indicate the centrosomes shown in merged inset). (B and C) Illustrative confocal images of COS cells expressing portions of GMAP-210 fused to GFP and costained with GM130 as in A, along with a summary of the results from all GMAP-210 portions examined.

Mentions: As described in the Specific residues within the Rud3p COOH terminus are critical for its localization, the GRAB domain identified in Rud3p is also present at the COOH terminus of the mammalian protein GMAP-210. This protein has been reported independently by two groups to be Golgi localized, although there is conflicting data as to how it interacts with the Golgi membranes (Rios et al., 1994; Chen et al., 1999; Infante et al., 1999). Infante et al. (1999) incubated Golgi membranes with recombinant fragments of GMAP-210 fused to GST. They concluded that the main interaction with membranes is mediated by the NH2-terminal 375 amino acids, and reported that a fusion between GFP and this portion of the protein was targeted to the Golgi in vivo. In addition, the COOH-terminal 202 residues of GMAP-210, which includes the GRAB domain, was reported to be targeted to centrosomes in vivo, and to bind γ-tubulin in vitro (Infante et al., 1999; Rios et al., 2004). In contrast, Chen et al. (1999) used fluorescence microscopy to examine in vivo the location of a series of GMAP-210 deletions. They reported that the COOH-terminal 226 residues of GMAP-210 are targeted to the Golgi. To investigate this further, we expressed in COS cells GFP fusions to the NH2-terminal 372 residues or the COOH-terminal 223 residues of GMAP-210. Protein blotting showed that the two fusion proteins were intact and accumulated to comparable levels (unpublished data). Fig. 7 A shows that the NH2-terminal fragment was not detectably associated with the Golgi, and instead fluorescence was observed throughout the cytoplasm. In contrast, the COOH-terminal domain localized to a juxtanuclear structure, which colocalized with the Golgi protein GM130, but did not colocalize with the centrosomal marker γ-tubulin (Fig. 7 A). It has recently been reported that overexpression of full-length GMAP-210 in COS cells results in accumulation of γ-tubulin on Golgi membranes and that this depends on the COOH-terminal 202 residues of the protein. However, in COS cells expressing either the COOH-terminal 223 residues or full-length GMAP-210, we did not observe γ-tubulin colocalizing with the protein on the Golgi (Fig. 7 A), even when the level of the full-length protein was so high that it induced Golgi fragmentation as previously reported (Pernet-Gallay et al., 2002). Therefore, our results indicate that the COOH terminus of GMAP-210 is responsible for Golgi rather than centrosomal targeting, in agreement with Chen et al. (1999).


The GTPase Arf1p and the ER to Golgi cargo receptor Erv14p cooperate to recruit the golgin Rud3p to the cis-Golgi.

Gillingham AK, Tong AH, Boone C, Munro S - J. Cell Biol. (2004)

The COOH-terminal region of human GMAP-210 mediates targeting to the Golgi rather than centrosomes. (A) Confocal micrographs of COS cells expressing the NH2-terminal 372 amino acids of GMAP-210 COOH-terminally tagged with GFP, or the COOH-terminal 223 amino acids of GMAP-210 NH2-terminally tagged with GFP, or full-length GMAP-210 with an NH2-terminal myc tag. Cells were also labeled with antibodies against the endogenous Golgi protein GM130 or γ-tubulin. At very high levels of GMAP-210, normal Golgi morphology (arrow) is lost and the Golgi becomes fragmented as described previously (Pernet-Gallay et al., 2002), and yet γ-tubulin is still only on the centrosome (arrowheads indicate the centrosomes shown in merged inset). (B and C) Illustrative confocal images of COS cells expressing portions of GMAP-210 fused to GFP and costained with GM130 as in A, along with a summary of the results from all GMAP-210 portions examined.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172552&req=5

fig7: The COOH-terminal region of human GMAP-210 mediates targeting to the Golgi rather than centrosomes. (A) Confocal micrographs of COS cells expressing the NH2-terminal 372 amino acids of GMAP-210 COOH-terminally tagged with GFP, or the COOH-terminal 223 amino acids of GMAP-210 NH2-terminally tagged with GFP, or full-length GMAP-210 with an NH2-terminal myc tag. Cells were also labeled with antibodies against the endogenous Golgi protein GM130 or γ-tubulin. At very high levels of GMAP-210, normal Golgi morphology (arrow) is lost and the Golgi becomes fragmented as described previously (Pernet-Gallay et al., 2002), and yet γ-tubulin is still only on the centrosome (arrowheads indicate the centrosomes shown in merged inset). (B and C) Illustrative confocal images of COS cells expressing portions of GMAP-210 fused to GFP and costained with GM130 as in A, along with a summary of the results from all GMAP-210 portions examined.
Mentions: As described in the Specific residues within the Rud3p COOH terminus are critical for its localization, the GRAB domain identified in Rud3p is also present at the COOH terminus of the mammalian protein GMAP-210. This protein has been reported independently by two groups to be Golgi localized, although there is conflicting data as to how it interacts with the Golgi membranes (Rios et al., 1994; Chen et al., 1999; Infante et al., 1999). Infante et al. (1999) incubated Golgi membranes with recombinant fragments of GMAP-210 fused to GST. They concluded that the main interaction with membranes is mediated by the NH2-terminal 375 amino acids, and reported that a fusion between GFP and this portion of the protein was targeted to the Golgi in vivo. In addition, the COOH-terminal 202 residues of GMAP-210, which includes the GRAB domain, was reported to be targeted to centrosomes in vivo, and to bind γ-tubulin in vitro (Infante et al., 1999; Rios et al., 2004). In contrast, Chen et al. (1999) used fluorescence microscopy to examine in vivo the location of a series of GMAP-210 deletions. They reported that the COOH-terminal 226 residues of GMAP-210 are targeted to the Golgi. To investigate this further, we expressed in COS cells GFP fusions to the NH2-terminal 372 residues or the COOH-terminal 223 residues of GMAP-210. Protein blotting showed that the two fusion proteins were intact and accumulated to comparable levels (unpublished data). Fig. 7 A shows that the NH2-terminal fragment was not detectably associated with the Golgi, and instead fluorescence was observed throughout the cytoplasm. In contrast, the COOH-terminal domain localized to a juxtanuclear structure, which colocalized with the Golgi protein GM130, but did not colocalize with the centrosomal marker γ-tubulin (Fig. 7 A). It has recently been reported that overexpression of full-length GMAP-210 in COS cells results in accumulation of γ-tubulin on Golgi membranes and that this depends on the COOH-terminal 202 residues of the protein. However, in COS cells expressing either the COOH-terminal 223 residues or full-length GMAP-210, we did not observe γ-tubulin colocalizing with the protein on the Golgi (Fig. 7 A), even when the level of the full-length protein was so high that it induced Golgi fragmentation as previously reported (Pernet-Gallay et al., 2002). Therefore, our results indicate that the COOH terminus of GMAP-210 is responsible for Golgi rather than centrosomal targeting, in agreement with Chen et al. (1999).

Bottom Line: Bornens. 2004.Cell. 118:323-335).In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge CB2 2QH, England, UK.

ABSTRACT
Rud3p is a coiled-coil protein of the yeast cis-Golgi. We find that Rud3p is localized to the Golgi via a COOH-terminal domain that is distantly related to the GRIP domain that recruits several coiled-coil proteins to the trans-Golgi by binding the small Arf-like GTPase Arl1p. In contrast, Rud3p binds to the GTPase Arf1p via this COOH-terminal "GRIP-related Arf-binding" (GRAB) domain. Deletion of RUD3 is lethal in the absence of the Golgi GTPase Ypt6p, and a screen of other mutants showing a similar genetic interaction revealed that Golgi targeting of Rud3p also requires Erv14p, a cargo receptor that cycles between the endoplasmic reticulum and Golgi. The one human protein with a GRAB domain, GMAP-210 (CEV14/Trip11/Trip230), is known to be on the cis-Golgi, but the COOH-terminal region that contains the GRAB domain has been reported to bind to centrosomes and gamma-tubulin (Rios, R.M, A. Sanchis, A.M. Tassin, C. Fedriani, and M. Bornens. 2004. Cell. 118:323-335). In contrast, we find that this region binds to the Golgi in a GRAB domain-dependent manner, suggesting that GMAP-210 may not link the Golgi to gamma-tubulin and centrosomes.

Show MeSH
Related in: MedlinePlus