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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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The overlapping activity of Gcs1 and Age2 is necessary for transport of the α-factor receptor, Ste3, to the vacuole. (A) Cells growing at 26°C were incubated at 37°C for 30 min, at which time cycloheximide was added, and were then either harvested immediately (0 min) or incubated at 37°C for a further 90 min. Cell extracts were resolved by SDS-PAGE and Ste3 protein was visualized by Western blot analysis. (B) Cells growing at 26°C were incubated at 37°C in medium containing cycloheximide. Portions of samples taken immediately or after a 120-min incubation were treated with Pronase to degrade cell surface proteins before Western blot analysis. The upper arrow indicates the position of intact Ste3, and the lower arrow indicates the position of Pronase-mediated breakdown products.
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fig6: The overlapping activity of Gcs1 and Age2 is necessary for transport of the α-factor receptor, Ste3, to the vacuole. (A) Cells growing at 26°C were incubated at 37°C for 30 min, at which time cycloheximide was added, and were then either harvested immediately (0 min) or incubated at 37°C for a further 90 min. Cell extracts were resolved by SDS-PAGE and Ste3 protein was visualized by Western blot analysis. (B) Cells growing at 26°C were incubated at 37°C in medium containing cycloheximide. Portions of samples taken immediately or after a 120-min incubation were treated with Pronase to degrade cell surface proteins before Western blot analysis. The upper arrow indicates the position of intact Ste3, and the lower arrow indicates the position of Pronase-mediated breakdown products.

Mentions: As a biochemical measure of endocytosis, we assessed the internalization and degradation of the α-factor mating–pheromone receptor Ste3. In wild-type cells, the Ste3 protein is constitutively removed from the plasma membrane, and is delivered via the endocytic pathway to the vacuole where it is degraded (Davis et al., 1993). In our assessment, cells were shifted to 37°C and incubated in the presence of the protein synthesis inhibitor cycloheximide to prevent further synthesis of Ste3 protein. As shown in Fig. 6 A, wild-type and single-mutant cells treated in this way showed a rapid loss of Ste3 protein, whereas in double-mutant cells the Ste3 protein was stable. The increased stability of Ste3 in double-mutant cells could indicate that the Ste3 protein was not internalized, or that Ste3 was internalized but not delivered to the vacuole for degradation. To resolve this issue, we incubated cells as above and then treated them with Pronase, a mixture of protein-degrading enzymes. Ste3 protein that remained at the plasma membrane would be exposed to enzymatic degradation, whereas Ste3 protein that had been successfully internalized but had failed to reach the vacuole would be expected to remain intact. As shown in Fig. 6 B, the Ste3 protein of double-mutant cells was resistant to Pronase digestion. We interpret this finding to indicate that Gcs1 + Age2 activity is not necessary for initial stages of endocytosis, but is required for effective delivery of endosomal cargo to the vacuole. These results are consistent with the internalization but cytoplasmic pattern of staining of membrane-bound FM4-64 in double-mutant cells, and suggest that Gcs1 + Age2 activity mediates effective endosomal transport after the internalization stage.


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)

The overlapping activity of Gcs1 and Age2 is necessary for transport of the α-factor receptor, Ste3, to the vacuole. (A) Cells growing at 26°C were incubated at 37°C for 30 min, at which time cycloheximide was added, and were then either harvested immediately (0 min) or incubated at 37°C for a further 90 min. Cell extracts were resolved by SDS-PAGE and Ste3 protein was visualized by Western blot analysis. (B) Cells growing at 26°C were incubated at 37°C in medium containing cycloheximide. Portions of samples taken immediately or after a 120-min incubation were treated with Pronase to degrade cell surface proteins before Western blot analysis. The upper arrow indicates the position of intact Ste3, and the lower arrow indicates the position of Pronase-mediated breakdown products.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: The overlapping activity of Gcs1 and Age2 is necessary for transport of the α-factor receptor, Ste3, to the vacuole. (A) Cells growing at 26°C were incubated at 37°C for 30 min, at which time cycloheximide was added, and were then either harvested immediately (0 min) or incubated at 37°C for a further 90 min. Cell extracts were resolved by SDS-PAGE and Ste3 protein was visualized by Western blot analysis. (B) Cells growing at 26°C were incubated at 37°C in medium containing cycloheximide. Portions of samples taken immediately or after a 120-min incubation were treated with Pronase to degrade cell surface proteins before Western blot analysis. The upper arrow indicates the position of intact Ste3, and the lower arrow indicates the position of Pronase-mediated breakdown products.
Mentions: As a biochemical measure of endocytosis, we assessed the internalization and degradation of the α-factor mating–pheromone receptor Ste3. In wild-type cells, the Ste3 protein is constitutively removed from the plasma membrane, and is delivered via the endocytic pathway to the vacuole where it is degraded (Davis et al., 1993). In our assessment, cells were shifted to 37°C and incubated in the presence of the protein synthesis inhibitor cycloheximide to prevent further synthesis of Ste3 protein. As shown in Fig. 6 A, wild-type and single-mutant cells treated in this way showed a rapid loss of Ste3 protein, whereas in double-mutant cells the Ste3 protein was stable. The increased stability of Ste3 in double-mutant cells could indicate that the Ste3 protein was not internalized, or that Ste3 was internalized but not delivered to the vacuole for degradation. To resolve this issue, we incubated cells as above and then treated them with Pronase, a mixture of protein-degrading enzymes. Ste3 protein that remained at the plasma membrane would be exposed to enzymatic degradation, whereas Ste3 protein that had been successfully internalized but had failed to reach the vacuole would be expected to remain intact. As shown in Fig. 6 B, the Ste3 protein of double-mutant cells was resistant to Pronase digestion. We interpret this finding to indicate that Gcs1 + Age2 activity is not necessary for initial stages of endocytosis, but is required for effective delivery of endosomal cargo to the vacuole. These results are consistent with the internalization but cytoplasmic pattern of staining of membrane-bound FM4-64 in double-mutant cells, and suggest that Gcs1 + Age2 activity mediates effective endosomal transport after the internalization stage.

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