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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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Absence of both Gcs1 and Age2 proteins is lethal. (A) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain was sporulated, and haploid segregants from resulting asci were incubated on solid medium at 30°C to assess growth. Segregant colonies were also replica plated to selective media to assess genotypes. Arrows indicate the positions of gcs1::URA3 age2::HIS3 double-mutant segregants. (B) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain containing plasmid pPP805-3, bearing the temperature-sensitive gcs1-3 allele, was sporulated, and the resulting segregants were assessed for growth at 30 and 37°C. Relevant genotypes are indicated.
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fig1: Absence of both Gcs1 and Age2 proteins is lethal. (A) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain was sporulated, and haploid segregants from resulting asci were incubated on solid medium at 30°C to assess growth. Segregant colonies were also replica plated to selective media to assess genotypes. Arrows indicate the positions of gcs1::URA3 age2::HIS3 double-mutant segregants. (B) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain containing plasmid pPP805-3, bearing the temperature-sensitive gcs1-3 allele, was sporulated, and the resulting segregants were assessed for growth at 30 and 37°C. Relevant genotypes are indicated.

Mentions: The yeast genome encodes four proteins with structural similarity to the Gcs1 and Glo3 ArfGAPs. The individual deletion of each of the genes encoding these proteins has little effect on cell growth (Zhang et al., 1998, and unpublished data). However, pair-wise deletions reveal that, like the deletion of both GCS1 and GLO3 genes, the deletion of GCS1 and the YIL044c open reading frame (referred to as SAT2 in Zhang et al., 1998) is lethal (Zhang et al., 1998, and Fig. 1 A). Therefore, we investigated the potentially overlapping functions provided by Gcs1 and the protein encoded by YIL044c, which has been assigned the name Age2 (ArfGAP effector).


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)

Absence of both Gcs1 and Age2 proteins is lethal. (A) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain was sporulated, and haploid segregants from resulting asci were incubated on solid medium at 30°C to assess growth. Segregant colonies were also replica plated to selective media to assess genotypes. Arrows indicate the positions of gcs1::URA3 age2::HIS3 double-mutant segregants. (B) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain containing plasmid pPP805-3, bearing the temperature-sensitive gcs1-3 allele, was sporulated, and the resulting segregants were assessed for growth at 30 and 37°C. Relevant genotypes are indicated.
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Absence of both Gcs1 and Age2 proteins is lethal. (A) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain was sporulated, and haploid segregants from resulting asci were incubated on solid medium at 30°C to assess growth. Segregant colonies were also replica plated to selective media to assess genotypes. Arrows indicate the positions of gcs1::URA3 age2::HIS3 double-mutant segregants. (B) A GCS1/gcs1::URA3 AGE2/ age2::HIS3 heterozygous diploid strain containing plasmid pPP805-3, bearing the temperature-sensitive gcs1-3 allele, was sporulated, and the resulting segregants were assessed for growth at 30 and 37°C. Relevant genotypes are indicated.
Mentions: The yeast genome encodes four proteins with structural similarity to the Gcs1 and Glo3 ArfGAPs. The individual deletion of each of the genes encoding these proteins has little effect on cell growth (Zhang et al., 1998, and unpublished data). However, pair-wise deletions reveal that, like the deletion of both GCS1 and GLO3 genes, the deletion of GCS1 and the YIL044c open reading frame (referred to as SAT2 in Zhang et al., 1998) is lethal (Zhang et al., 1998, and Fig. 1 A). Therefore, we investigated the potentially overlapping functions provided by Gcs1 and the protein encoded by YIL044c, which has been assigned the name Age2 (ArfGAP effector).

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