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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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COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3, but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN, URA3 plasmid were transformed with CEN, LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.
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fig4: COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3, but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN, URA3 plasmid were transformed with CEN, LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.

Mentions: To examine this further, a strain (AGY24) was created in which RUD3 and RIC1 were deleted from the genome, with viability maintained by a CEN plasmid expressing Ric1p. When GFP-Rud3p was expressed in this strain it was able to maintain growth upon loss of the RIC1 plasmid, indicating that the fusion protein is functional. However, GFP-Rud3p (L410A) could not sustain growth in the absence of Ric1p (Fig. 4 A). In addition, mutation of another conserved residue, D407A, resulted in slow growth after loss of Ric1p, with this mutation causing a partial delocalization from the Golgi (unpublished data). These data indicate that the GRAB domain is required not only for Rud3p targeting but also for its function.


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)

COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3, but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN, URA3 plasmid were transformed with CEN, LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.
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fig4: COOH-terminal mutants of Rud3p are not functional. (A) Growth at the indicated temperatures of yeast lacking genomic copies of both RIC1 and RUD3, but containing the either an empty plasmid or expressing wild-type or mutant GFP-Rud3p (indicated on the diagram) from a constitutive PHO5 promoter. Cells lacking RUD3 and with only copy of RIC1 on a CEN, URA3 plasmid were transformed with CEN, LEU2 plasmids expressing GFP-Rud3p or mutants, and the yeast plated onto plates containing 5-fluoroorotic acid (5-FOA) to remove the RIC1 containing URA3 plasmid. (B) A yeast strain expressing GFP-tagged Rud3p lacking the GA1 motif (463ΔC) was used in a binding assay as in Fig. 3 C with GST-Arf1p loaded with either GDP or GTP-γ-S. Also shown is a fluorescent micrograph of live yeast lacking genomic RUD3 and expressing GFP-Rud3p463ΔC. (C) Fluorescent micrographs of a rud3Δ strain with the ARF1 ORF tagged in the genome with a COOH-terminal GFP tag (AGY26) and expressing RFP-Rud3p under the constitutive PHO5 promoter from the CEN plasmid pRS416. Some structures contain both RFP-Rud3p and Arf1p-GFP (arrows) and some contain just the latter (arrowheads). (D) Fluorescent micrographs of live BY4741 yeast with the indicated genes deleted and expressing GFP-Rud3p as in Fig. 1 C.
Mentions: To examine this further, a strain (AGY24) was created in which RUD3 and RIC1 were deleted from the genome, with viability maintained by a CEN plasmid expressing Ric1p. When GFP-Rud3p was expressed in this strain it was able to maintain growth upon loss of the RIC1 plasmid, indicating that the fusion protein is functional. However, GFP-Rud3p (L410A) could not sustain growth in the absence of Ric1p (Fig. 4 A). In addition, mutation of another conserved residue, D407A, resulted in slow growth after loss of Ric1p, with this mutation causing a partial delocalization from the Golgi (unpublished data). These data indicate that the GRAB domain is required not only for Rud3p targeting but also for its function.

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