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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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Secretion of invertase is impaired by inadequate Gcs1 + Age2 activity. Cells growing at 26°C were transferred to low-glucose medium to induce invertase expression and incubated at 37°C for 45 min. Secreted and intracellular invertase was separated into internal (i) and external (e) fractions and resolved by SDS-PAGE without heat treating the samples. Invertase was detected by an in-gel enzyme assay (Gabriel and Wang, 1969). For reference, a sec18 mutant was incubated at 38°C before invertase induction as above. The arrow indicates the position of the ER-glycosylated form of invertase. Longer incubations of up to 2 h did not change the pattern or distribution of invertase in mutant cells (unpublished data).
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fig3: Secretion of invertase is impaired by inadequate Gcs1 + Age2 activity. Cells growing at 26°C were transferred to low-glucose medium to induce invertase expression and incubated at 37°C for 45 min. Secreted and intracellular invertase was separated into internal (i) and external (e) fractions and resolved by SDS-PAGE without heat treating the samples. Invertase was detected by an in-gel enzyme assay (Gabriel and Wang, 1969). For reference, a sec18 mutant was incubated at 38°C before invertase induction as above. The arrow indicates the position of the ER-glycosylated form of invertase. Longer incubations of up to 2 h did not change the pattern or distribution of invertase in mutant cells (unpublished data).

Mentions: As an initial indicator of vesicular transport that may be affected by Gcs1 + Age2 activity, we assessed the glycosylation status of the extracellular enzyme invertase and the effectiveness of invertase secretion. Invertase expression is induced in low-glucose medium, and newly synthesized invertase undergoes N-linked glycosylation in the ER. These invertase molecules are then transported to the Golgi, where further glycosylation ensues; the extent of glycosylation is reflected in the degree of decrease in invertase mobility during gel electrophoresis. Growing cells were transferred to low-glucose medium to induce the synthesis of invertase, and were incubated at 37°C. For wild-type cells and in gcs1-3 and age2 single-mutant cells, invertase was found predominantly in the external fraction, in highly glycosylated form (Fig. 3). In sec18 mutant control cells, defective for transport out of the ER (Novick et al., 1980), invertase was only core glycosylated and was not secreted (Fig. 3). In marked contrast, gcs1-3 age2 double-mutant cells had invertase in a highly glycosylated form, indicating exposure to Golgi-resident enzymes (Fig. 3). However, about half of this glycosylated invertase remained inside the cells, indicating that transport from the Golgi to the plasma membrane is impaired.


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)

Secretion of invertase is impaired by inadequate Gcs1 + Age2 activity. Cells growing at 26°C were transferred to low-glucose medium to induce invertase expression and incubated at 37°C for 45 min. Secreted and intracellular invertase was separated into internal (i) and external (e) fractions and resolved by SDS-PAGE without heat treating the samples. Invertase was detected by an in-gel enzyme assay (Gabriel and Wang, 1969). For reference, a sec18 mutant was incubated at 38°C before invertase induction as above. The arrow indicates the position of the ER-glycosylated form of invertase. Longer incubations of up to 2 h did not change the pattern or distribution of invertase in mutant cells (unpublished data).
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Related In: Results  -  Collection

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fig3: Secretion of invertase is impaired by inadequate Gcs1 + Age2 activity. Cells growing at 26°C were transferred to low-glucose medium to induce invertase expression and incubated at 37°C for 45 min. Secreted and intracellular invertase was separated into internal (i) and external (e) fractions and resolved by SDS-PAGE without heat treating the samples. Invertase was detected by an in-gel enzyme assay (Gabriel and Wang, 1969). For reference, a sec18 mutant was incubated at 38°C before invertase induction as above. The arrow indicates the position of the ER-glycosylated form of invertase. Longer incubations of up to 2 h did not change the pattern or distribution of invertase in mutant cells (unpublished data).
Mentions: As an initial indicator of vesicular transport that may be affected by Gcs1 + Age2 activity, we assessed the glycosylation status of the extracellular enzyme invertase and the effectiveness of invertase secretion. Invertase expression is induced in low-glucose medium, and newly synthesized invertase undergoes N-linked glycosylation in the ER. These invertase molecules are then transported to the Golgi, where further glycosylation ensues; the extent of glycosylation is reflected in the degree of decrease in invertase mobility during gel electrophoresis. Growing cells were transferred to low-glucose medium to induce the synthesis of invertase, and were incubated at 37°C. For wild-type cells and in gcs1-3 and age2 single-mutant cells, invertase was found predominantly in the external fraction, in highly glycosylated form (Fig. 3). In sec18 mutant control cells, defective for transport out of the ER (Novick et al., 1980), invertase was only core glycosylated and was not secreted (Fig. 3). In marked contrast, gcs1-3 age2 double-mutant cells had invertase in a highly glycosylated form, indicating exposure to Golgi-resident enzymes (Fig. 3). However, about half of this glycosylated invertase remained inside the cells, indicating that transport from the Golgi to the plasma membrane is impaired.

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