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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 terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 (Robinson et al., 1988) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH2-terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p (Lupas, 1996). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.
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fig1: The COOH terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 (Robinson et al., 1988) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH2-terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p (Lupas, 1996). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.

Mentions: To investigate how Rud3p is recruited to the Golgi, we initially expressed the full-length protein tagged at the NH2 terminus with a nonmultimerizing form of DsRed (“RFP,” tdimer2(12); Campbell et al., 2002). RFP-Rud3p accumulated in punctate structures that colocalized with a GFP-tagged form of the vesicle tethering protein Uso1p (Fig. 1 A). Uso1p apparently tethers ER-derived vesicles to Golgi membranes, and hence is a marker for the earliest, or cis, compartment of the Golgi (Cao et al., 1998). To map the region of Rud3p required for this cis-Golgi targeting, a GFP cassette was introduced at a series of locations through the genomic copy of the RUD3 gene (Fig. 1 B). This approach allows targeting to be examined in the absence of full-length endogenous protein. Removal of the NH2-terminal 194 of the 484 residues of Rud3p did not detectably affect Rud3p targeting (Fig. 1 C). Further NH2-terminal truncation led to a partial cytoplasmic distribution of the tagged protein. However, protein blotting indicated that this relocalization was most likely due to increased protein instability (unpublished data). Nonetheless, detectable membrane targeting was still observed with GFP fused to residue 405 of Rud3p (405–484; Fig. 1 C). These results indicate that Rud3p is recruited to the cis-Golgi, and that targeting information is encoded within the COOH-terminal 80 residues of the protein.


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 terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 (Robinson et al., 1988) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH2-terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p (Lupas, 1996). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172552&req=5

fig1: The COOH terminus of Rud3p mediates Golgi association. (A) Fluorescent micrographs of live yeast expressing the indicated fusion proteins. Uso1p was tagged with GFP at the COOH terminus in the genome of the wild-type strain BY4741, whereas RFP-Rud3p was expressed from a CEN plasmid under the control of a constitutive version of the PHO5 promoter. The two proteins were found to be localized to the same punctate structures, as illustrated by those marked with arrows. (B) Schematic diagram of truncations made in the RUD3 ORF in the yeast strain SEY6210 (Robinson et al., 1988) by insertion by homologous recombination of a cassette encoding a PHO5 promoter and an NH2-terminal GFP tag. Also shown is a coiled-coil prediction for Rud3p (Lupas, 1996). (C) Fluorescent micrographs of live yeast expressing the indicated GFP-Rud3p truncations as in B.
Mentions: To investigate how Rud3p is recruited to the Golgi, we initially expressed the full-length protein tagged at the NH2 terminus with a nonmultimerizing form of DsRed (“RFP,” tdimer2(12); Campbell et al., 2002). RFP-Rud3p accumulated in punctate structures that colocalized with a GFP-tagged form of the vesicle tethering protein Uso1p (Fig. 1 A). Uso1p apparently tethers ER-derived vesicles to Golgi membranes, and hence is a marker for the earliest, or cis, compartment of the Golgi (Cao et al., 1998). To map the region of Rud3p required for this cis-Golgi targeting, a GFP cassette was introduced at a series of locations through the genomic copy of the RUD3 gene (Fig. 1 B). This approach allows targeting to be examined in the absence of full-length endogenous protein. Removal of the NH2-terminal 194 of the 484 residues of Rud3p did not detectably affect Rud3p targeting (Fig. 1 C). Further NH2-terminal truncation led to a partial cytoplasmic distribution of the tagged protein. However, protein blotting indicated that this relocalization was most likely due to increased protein instability (unpublished data). Nonetheless, detectable membrane targeting was still observed with GFP fused to residue 405 of Rud3p (405–484; Fig. 1 C). These results indicate that Rud3p is recruited to the cis-Golgi, and that targeting information is encoded within the COOH-terminal 80 residues of the protein.

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