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Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M, Nilsson T - J. Cell Biol. (2001)

Bottom Line: Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride.Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes.We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69017 Heidelberg, Germany.

ABSTRACT
We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

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A requirement for GTP hydrolysis by Arf-1. (A) Vesicles were formed in the presence of GTP, GTPγS, or Arf-1Q71L. (B) Vesicles were formed in the presence of GTP, GTPγS, BeFx, or AlFx. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, β-COP, p24γ3, p24α2, p24δ1, GS28, p24β1, and the KDR followed by ECL.
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fig4: A requirement for GTP hydrolysis by Arf-1. (A) Vesicles were formed in the presence of GTP, GTPγS, or Arf-1Q71L. (B) Vesicles were formed in the presence of GTP, GTPγS, BeFx, or AlFx. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, β-COP, p24γ3, p24α2, p24δ1, GS28, p24β1, and the KDR followed by ECL.

Mentions: We showed previously that Mann II and two glycosyltransferases, N-acetylglucosaminyltransferase I (GlcNAc-T1) and galactosyltransferase (GT-1), are sorted and concentrated into COPI vesicles in an Arf-1 GTP hydrolysis–dependent manner (Lanoix et al., 1999). Yet, resident proteins such as p24 proteins are proposed to inhibit GTP hydrolysis, thereby ensuring that coatomer can remain on the membrane in order to form “priming complexes” required for vesicle formation (Springer et al., 1999). To investigate this, we first tested whether the four p24 proteins require GTP hydrolysis by Arf-1 for incorporation into vesicles. As seen in Fig. 4 A, we observed a clear decrease for p24α2, β1, δ1, and γ3 upon addition of GTPγS, a nonhydrolyzable analogue of GTP or the GTP-restricted mutant of Arf-1, ArfQ71L. A similar inhibition, though less striking, was observed upon probing for the KDEL receptor (KDR). As expected, coatomer was most readily detected (monitored using monoclonal antibodies to β-COP) on vesicles generated under conditions where GTP hydrolysis was inhibited. However, a small amount of coatomer persisted on vesicles generated in the presence of GTP. This was observed consistently using our modified assay, suggesting that perhaps uncoating efficiency was somewhat lowered.


Sorting of Golgi resident proteins into different subpopulations of COPI vesicles: a role for ArfGAP1.

Lanoix J, Ouwendijk J, Stark A, Szafer E, Cassel D, Dejgaard K, Weiss M, Nilsson T - J. Cell Biol. (2001)

A requirement for GTP hydrolysis by Arf-1. (A) Vesicles were formed in the presence of GTP, GTPγS, or Arf-1Q71L. (B) Vesicles were formed in the presence of GTP, GTPγS, BeFx, or AlFx. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, β-COP, p24γ3, p24α2, p24δ1, GS28, p24β1, and the KDR followed by ECL.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199348&req=5

fig4: A requirement for GTP hydrolysis by Arf-1. (A) Vesicles were formed in the presence of GTP, GTPγS, or Arf-1Q71L. (B) Vesicles were formed in the presence of GTP, GTPγS, BeFx, or AlFx. Proteins from solubilized membranes or vesicles were separated by SDS-PAGE and subjected to Western blot analysis using specific primary antibodies to Mann II, β-COP, p24γ3, p24α2, p24δ1, GS28, p24β1, and the KDR followed by ECL.
Mentions: We showed previously that Mann II and two glycosyltransferases, N-acetylglucosaminyltransferase I (GlcNAc-T1) and galactosyltransferase (GT-1), are sorted and concentrated into COPI vesicles in an Arf-1 GTP hydrolysis–dependent manner (Lanoix et al., 1999). Yet, resident proteins such as p24 proteins are proposed to inhibit GTP hydrolysis, thereby ensuring that coatomer can remain on the membrane in order to form “priming complexes” required for vesicle formation (Springer et al., 1999). To investigate this, we first tested whether the four p24 proteins require GTP hydrolysis by Arf-1 for incorporation into vesicles. As seen in Fig. 4 A, we observed a clear decrease for p24α2, β1, δ1, and γ3 upon addition of GTPγS, a nonhydrolyzable analogue of GTP or the GTP-restricted mutant of Arf-1, ArfQ71L. A similar inhibition, though less striking, was observed upon probing for the KDEL receptor (KDR). As expected, coatomer was most readily detected (monitored using monoclonal antibodies to β-COP) on vesicles generated under conditions where GTP hydrolysis was inhibited. However, a small amount of coatomer persisted on vesicles generated in the presence of GTP. This was observed consistently using our modified assay, suggesting that perhaps uncoating efficiency was somewhat lowered.

Bottom Line: Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride.Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes.We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Biophysics Programme, European Molecular Biology Laboratory, D-69017 Heidelberg, Germany.

ABSTRACT
We present evidence for two subpopulations of coatomer protein I vesicles, both containing high amounts of Golgi resident proteins but only minor amounts of anterograde cargo. Early Golgi proteins p24alpha2, beta1, delta1, and gamma3 are shown to be sorted together into vesicles that are distinct from those containing mannosidase II, a glycosidase of the medial Golgi stack, and GS28, a SNARE protein of the Golgi stack. Sorting into each vesicle population is Arf-1 and GTP hydrolysis dependent and is inhibited by aluminum and beryllium fluoride. Using synthetic peptides, we find that the cytoplasmic domain of p24beta1 can bind Arf GTPase-activating protein (GAP)1 and cause direct inhibition of ArfGAP1-mediated GTP hydrolysis on Arf-1 bound to liposomes and Golgi membranes. We propose a two-stage reaction to explain how GTP hydrolysis constitutes a prerequisite for sorting of resident proteins, yet becomes inhibited in their presence.

Show MeSH
Related in: MedlinePlus