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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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Cells with inadequate Gcs1 + Age2 activity are impaired for endocytosis. Cells growing at 23°C were incubated with the membrane dye FM4-64 for 10 min, transferred to fresh dye-free medium for a further 1-h incubation at 23 or 37°C, and visualized by DIC optics and fluorescence microscopy (FM4-64).
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fig5: Cells with inadequate Gcs1 + Age2 activity are impaired for endocytosis. Cells growing at 23°C were incubated with the membrane dye FM4-64 for 10 min, transferred to fresh dye-free medium for a further 1-h incubation at 23 or 37°C, and visualized by DIC optics and fluorescence microscopy (FM4-64).

Mentions: To assess whether endosomal trafficking is affected in gcs1-3 age2 double-mutant cells, we determined the ability of cells to transport the lipophilic dye FM4-64 from the plasma membrane to the vacuole (Vida and Emr, 1995), a transport process that involves routing of membrane-bound FM4-64 through the endosomal compartment. Wild-type cells (Fig. 5) and single-mutant cells (unpublished data) at 37°C showed a significant localization of FM4-64 to the vacuole, as indicated by a high concentration of the dye outlining the vacuolar membrane. In marked contrast, although gcs1-3 age2 double-mutant cells were able to internalize the dye, they displayed a diffuse and granular cytoplasmic staining pattern, with indistinct vacuolar membrane staining (Fig. 5). Even after extended incubation, mutant cells remained impaired in the delivery of the FM4-64 dye to the vacuole (unpublished data). Thus, inadequate Gcs1 + Age2 activity also impairs endocytosis, suggesting that the Gcs1 + Age2 proteins provide overlapping function for vesicular transport affecting endosomal traffic.


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)

Cells with inadequate Gcs1 + Age2 activity are impaired for endocytosis. Cells growing at 23°C were incubated with the membrane dye FM4-64 for 10 min, transferred to fresh dye-free medium for a further 1-h incubation at 23 or 37°C, and visualized by DIC optics and fluorescence microscopy (FM4-64).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Cells with inadequate Gcs1 + Age2 activity are impaired for endocytosis. Cells growing at 23°C were incubated with the membrane dye FM4-64 for 10 min, transferred to fresh dye-free medium for a further 1-h incubation at 23 or 37°C, and visualized by DIC optics and fluorescence microscopy (FM4-64).
Mentions: To assess whether endosomal trafficking is affected in gcs1-3 age2 double-mutant cells, we determined the ability of cells to transport the lipophilic dye FM4-64 from the plasma membrane to the vacuole (Vida and Emr, 1995), a transport process that involves routing of membrane-bound FM4-64 through the endosomal compartment. Wild-type cells (Fig. 5) and single-mutant cells (unpublished data) at 37°C showed a significant localization of FM4-64 to the vacuole, as indicated by a high concentration of the dye outlining the vacuolar membrane. In marked contrast, although gcs1-3 age2 double-mutant cells were able to internalize the dye, they displayed a diffuse and granular cytoplasmic staining pattern, with indistinct vacuolar membrane staining (Fig. 5). Even after extended incubation, mutant cells remained impaired in the delivery of the FM4-64 dye to the vacuole (unpublished data). Thus, inadequate Gcs1 + Age2 activity also impairs endocytosis, suggesting that the Gcs1 + Age2 proteins provide overlapping function for vesicular transport affecting endosomal traffic.

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