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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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Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN, URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.
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fig6: Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN, URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.

Mentions: The redistribution of Rud3p in cells lacking Erv14p is not due to mislocalization of Arf1p as the GTPase is not relocalized to the cytoplasm in an erv14Δ strain (Fig. 6 A). In addition, loss of Erv14p does not appear to cause an alteration in Rud3p that renders it incapable of binding Arf1p, as GFP-Rud3p from strains lacking Erv14p was still able to associate with Arf1p-GTP in vitro (Fig. 6 B). Finally, we examined whether or not Rud3p is required for Erv14p to function. Deletion of Erv14p causes the cargo protein Axl2p to accumulate in the ER, resulting in increased gel mobility due to the absence of Golgi processing of N- and O-linked glycans (Powers and Barlowe, 1998). However, in a rud3Δ strain the ER form of Axl2p did not accumulate, indicating that Rud3p is not required for the Erv14p-dependent ER exit of Axl2p (Fig. 6 C). However, in this strain the N-linked glycans on Axl2p were not as extensively modified as in wild-type, which is consistent with the partial defect in Golgi glycan modification previously reported with a different substrate (Kim, 2003). Fig. 6 D shows that this phenotype could not be rescued by Rud3p carrying the L410A mutant that prevents Arf1p binding, indicating that interaction between Rud3p and Arf1p is required for normal Golgi 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)

Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN, URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.
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fig6: Erv14p and Rud3p in Golgi function. (A) Fluorescent micrographs of yeast (BY4741) expressing Arf1p-GFP from a CEN, URA3 plasmid, with the genomic copies of ARF1 and ERV14 deleted as indicated. (B) Anti-GFP protein blot of lysates (Lys.; 10% of material loaded) from wild-type BY4741 (WT) or erv14Δ cells expressing GFP-Rud3p or of the proteins bound when the lysates were applied to immobilized GST-Arf1p loaded with the indicated nucleotides, as in Fig. 3 C. (C) Anti-GFP protein blot of total cellular proteins from BY4741 (WT) or the indicated strains expressing Axl2p-GFP from a CEN plasmid, with or without endoglycosidase H digestion. (D) As C, except that the cells were rud3Δ and contained either an empty CEN vector or the same with the indicated forms of GFP-Rud3p.
Mentions: The redistribution of Rud3p in cells lacking Erv14p is not due to mislocalization of Arf1p as the GTPase is not relocalized to the cytoplasm in an erv14Δ strain (Fig. 6 A). In addition, loss of Erv14p does not appear to cause an alteration in Rud3p that renders it incapable of binding Arf1p, as GFP-Rud3p from strains lacking Erv14p was still able to associate with Arf1p-GTP in vitro (Fig. 6 B). Finally, we examined whether or not Rud3p is required for Erv14p to function. Deletion of Erv14p causes the cargo protein Axl2p to accumulate in the ER, resulting in increased gel mobility due to the absence of Golgi processing of N- and O-linked glycans (Powers and Barlowe, 1998). However, in a rud3Δ strain the ER form of Axl2p did not accumulate, indicating that Rud3p is not required for the Erv14p-dependent ER exit of Axl2p (Fig. 6 C). However, in this strain the N-linked glycans on Axl2p were not as extensively modified as in wild-type, which is consistent with the partial defect in Golgi glycan modification previously reported with a different substrate (Kim, 2003). Fig. 6 D shows that this phenotype could not be rescued by Rud3p carrying the L410A mutant that prevents Arf1p binding, indicating that interaction between Rud3p and Arf1p is required for normal Golgi 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