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The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network.

Poon PP, Nothwehr SF, Singer RA, Johnston GC - J. Cell Biol. (2001)

Bottom Line: Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins.Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport.Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.

ABSTRACT
Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

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Related in: MedlinePlus

Age2 is an ArfGAP. GAP activity of recombinant Age2 protein (•) was assessed by in vitro hydrolysis of radiolabeled GTP bound to recombinant yeast Arf1 protein. For comparison, the activity of the Gcs1 ArfGAP (○) on Arf1 protein is also displayed. Arf-bound GTP was not hydrolysed when assayed with bovine serum albumin over the same protein concentrations (unpublished data). Activity is expressed as percent of maximum radioactivity released from GTP-Arf with the highest level of release expressed as 100%. Recombinant Gcs1 ArfGAP was able to stimulate hydrolysis of >90% of labeled material associated with recombinant Arf1 protein.
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fig2: Age2 is an ArfGAP. GAP activity of recombinant Age2 protein (•) was assessed by in vitro hydrolysis of radiolabeled GTP bound to recombinant yeast Arf1 protein. For comparison, the activity of the Gcs1 ArfGAP (○) on Arf1 protein is also displayed. Arf-bound GTP was not hydrolysed when assayed with bovine serum albumin over the same protein concentrations (unpublished data). Activity is expressed as percent of maximum radioactivity released from GTP-Arf with the highest level of release expressed as 100%. Recombinant Gcs1 ArfGAP was able to stimulate hydrolysis of >90% of labeled material associated with recombinant Arf1 protein.

Mentions: As with Gcs1 and Glo3, the most striking similarity between the Gcs1 and Age2 proteins is in an NH2-terminal 68-residue region of each protein, including the Zn-binding motif required for ArfGAP activity in vitro (Cukierman et al., 1995; Poon et al., 1996). To determine if the structural similarity among the Gcs1, Glo3, and Age2 proteins extends to function, we assessed the ArfGAP activity of Age2. Recombinant Age2 protein was produced in Escherichia coli and assessed in vitro using recombinant yeast Arf1 protein as substrate. As expected based on structural considerations, Age2 protein was able to stimulate the hydrolysis of Arf1-bound GTP to GDP (Fig. 2). Under the assay conditions used here, Age2 ArfGAP activity was approximately tenfold lower than that of Gcs1, similar to the ratio of activity of Glo3 to Gcs1 (Poon et al., 1999). Like Gcs1 and Glo3, the Age2 protein is a yeast ArfGAP.


The Gcs1 and Age2 ArfGAP proteins provide overlapping essential function for transport from the yeast trans-Golgi network.

Poon PP, Nothwehr SF, Singer RA, Johnston GC - J. Cell Biol. (2001)

Age2 is an ArfGAP. GAP activity of recombinant Age2 protein (•) was assessed by in vitro hydrolysis of radiolabeled GTP bound to recombinant yeast Arf1 protein. For comparison, the activity of the Gcs1 ArfGAP (○) on Arf1 protein is also displayed. Arf-bound GTP was not hydrolysed when assayed with bovine serum albumin over the same protein concentrations (unpublished data). Activity is expressed as percent of maximum radioactivity released from GTP-Arf with the highest level of release expressed as 100%. Recombinant Gcs1 ArfGAP was able to stimulate hydrolysis of >90% of labeled material associated with recombinant Arf1 protein.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199332&req=5

fig2: Age2 is an ArfGAP. GAP activity of recombinant Age2 protein (•) was assessed by in vitro hydrolysis of radiolabeled GTP bound to recombinant yeast Arf1 protein. For comparison, the activity of the Gcs1 ArfGAP (○) on Arf1 protein is also displayed. Arf-bound GTP was not hydrolysed when assayed with bovine serum albumin over the same protein concentrations (unpublished data). Activity is expressed as percent of maximum radioactivity released from GTP-Arf with the highest level of release expressed as 100%. Recombinant Gcs1 ArfGAP was able to stimulate hydrolysis of >90% of labeled material associated with recombinant Arf1 protein.
Mentions: As with Gcs1 and Glo3, the most striking similarity between the Gcs1 and Age2 proteins is in an NH2-terminal 68-residue region of each protein, including the Zn-binding motif required for ArfGAP activity in vitro (Cukierman et al., 1995; Poon et al., 1996). To determine if the structural similarity among the Gcs1, Glo3, and Age2 proteins extends to function, we assessed the ArfGAP activity of Age2. Recombinant Age2 protein was produced in Escherichia coli and assessed in vitro using recombinant yeast Arf1 protein as substrate. As expected based on structural considerations, Age2 protein was able to stimulate the hydrolysis of Arf1-bound GTP to GDP (Fig. 2). Under the assay conditions used here, Age2 ArfGAP activity was approximately tenfold lower than that of Gcs1, similar to the ratio of activity of Glo3 to Gcs1 (Poon et al., 1999). Like Gcs1 and Glo3, the Age2 protein is a yeast ArfGAP.

Bottom Line: Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins.Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport.Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.

ABSTRACT
Many intracellular vesicle transport pathways involve GTP hydrolysis by the ADP-ribosylation factor (ARF) type of monomeric G proteins, under the control of ArfGAP proteins. Here we show that the structurally related yeast proteins Gcs1 and Age2 form an essential ArfGAP pair that provides overlapping function for TGN transport. Mutant cells lacking the Age2 and Gcs1 proteins cease proliferation, accumulate membranous structures resembling Berkeley bodies, and are unable to properly process and localize the vacuolar hydrolase carboxypeptidase (CPY) and the vacuolar membrane protein alkaline phosphatase (ALP), which are transported from the TGN to the vacuole by distinct transport routes. Immunofluorescence studies localizing the proteins ALP, Kex2 (a TGN resident protein), and Vps10 (the CPY receptor for transport from the TGN to the vacuole) suggest that inadequate function of this ArfGAP pair leads to a fragmentation of TGN, with effects on secretion and endosomal transport. Our results demonstrate that the Gcs1 + Age2 ArfGAP pair provides overlapping function for transport from the TGN, and also indicate that multiple activities at the TGN can be maintained with the aid of a single ArfGAP.

Show MeSH
Related in: MedlinePlus