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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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Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH2-terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH2-terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides (Shevchenko et al., 1996).
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fig3: Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH2-terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH2-terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides (Shevchenko et al., 1996).

Mentions: GRIP domain proteins are recruited to the Golgi by interaction with the small GTP-binding protein Arl1. Because the GRIP and GRAB domains appear related, we examined whether or not Rud3p interacts with a small GTP-binding protein on the Golgi. In yeast there are seven small GTP-binding proteins that have been found on Golgi membranes, Arf1p, Arl1p, Arl3p, Ypt1p, Ypt6p, Ypt31p, and Ypt32p. In addition, Arf3p (the closest yeast relative of mammalian Arf6) was analyzed as its location was unknown at the time, although it was subsequently reported to be on the plasma membrane (Huang et al., 2003). Of these GTPases, Arl1p and Arl3p are unlikely candidates to bind Rud3p as they are localized to the late Golgi for the recruitment of the GRIP domain protein Imh1p (Gangi Setty et al., 2003; Panic et al., 2003a). Thus, the other six proteins were expressed in Escherichia coli as fusions to GST, with a Q to L mutation in the GTPase motif. This mutation has been found in other Ras family GTPases to lock the proteins in a GTP-bound state. The GST fusions were immobilized on beads and incubated with lysates from yeast expressing HA-tagged Rud3p. HA-Rud3p bound to both the Golgi-specific GTPase Arf1p and the plasma membrane GTPase Arf3p, but not to the other Golgi GTPases (Fig. 3 A).


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)

Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH2-terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH2-terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides (Shevchenko et al., 1996).
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Related In: Results  -  Collection

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fig3: Rud3p interacts with the small GTPase Arf1p. (A) Anti-HA protein blot of total cell lysate (Lys.) from a strain expressing Rud3p tagged in the genome with an NH2-terminal HA epitope tag (AGY10) and of proteins that bound to GST fusions of the GTP-locked versions of Arf1p, Arf3p, Ypt1p, Ypt6, Ypt31p, and Ypt32p. For the GTP-locked forms of Arf1p (Q71L) and Arf3p (Q71L), the first 14 amino acids that form an amphipathic helix were removed and replaced with an NH2-terminal GST tag. For Ypt1p (Q67L), Ypt6p (Q67L), Ypt31p (Q72L), and Ypt32p (Q72L), the COOH-terminal cysteine residues were replaced with a COOH-terminal GST tag. Lysate is 10% of material applied to beads. (B) Anti-HA protein blot of total cell lysate (Lys.; 10% of material loaded) and proteins that bound to GST fusions of wild-type Arf1p and Arf3p preloaded with GDP or a nonhydrolysable analogue of GTP, GTPγS. The blot was stripped and reprobed with a rabbit antibody against Imh1p. (C) As for GST-Arf1p in B, except cell lysates were prepared from a strain expressing either GFP-Rud3p or the mutant proteins L410A expressed under the control of a constitutively active PHO5 promoter from the CEN plasmid pRS416. (D) Binding of the COOH-terminal 126 amino acids of Rud3p to Arf1p (T31N) or Arf1p (Q71L). The indicated forms of GST-Arf1p were coexpressed in E. coli with the COOH terminus of Rud3p, and after cell lysis isolated on glutathione Sepharose beads. Bound proteins were analyzed by gel electrophoresis, and the indicated band was identified as the COOH terminus of Rud3p by matrix-assisted laser desorption ionization mass spectrometry of tryptic peptides (Shevchenko et al., 1996).
Mentions: GRIP domain proteins are recruited to the Golgi by interaction with the small GTP-binding protein Arl1. Because the GRIP and GRAB domains appear related, we examined whether or not Rud3p interacts with a small GTP-binding protein on the Golgi. In yeast there are seven small GTP-binding proteins that have been found on Golgi membranes, Arf1p, Arl1p, Arl3p, Ypt1p, Ypt6p, Ypt31p, and Ypt32p. In addition, Arf3p (the closest yeast relative of mammalian Arf6) was analyzed as its location was unknown at the time, although it was subsequently reported to be on the plasma membrane (Huang et al., 2003). Of these GTPases, Arl1p and Arl3p are unlikely candidates to bind Rud3p as they are localized to the late Golgi for the recruitment of the GRIP domain protein Imh1p (Gangi Setty et al., 2003; Panic et al., 2003a). Thus, the other six proteins were expressed in Escherichia coli as fusions to GST, with a Q to L mutation in the GTPase motif. This mutation has been found in other Ras family GTPases to lock the proteins in a GTP-bound state. The GST fusions were immobilized on beads and incubated with lysates from yeast expressing HA-tagged Rud3p. HA-Rud3p bound to both the Golgi-specific GTPase Arf1p and the plasma membrane GTPase Arf3p, but not to the other Golgi GTPases (Fig. 3 A).

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