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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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Transport of ALP to the vacuole is impaired in cells with inadequate Gcs1 + Age2 activity. (A) Cells growing at 26°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 7 min, and incubated further with unlabeled amino acids. ALP was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The precursor (P) and mature (M) forms are indicated. After 30 min, wild-type cells had >90% of ALP in the mature form, whereas double-mutant cells had 75% of ALP in the precursor form. (B) Cells carrying plasmid pSN351, harboring the ALP gene PHO8 expressed from the inducible GAL1 promoter, were grown at 23°C in raffinose medium and transferred to 37°C. After a 15-min incubation, ALP synthesis was induced by the addition of galactose. After a 40-min induction period, glucose was added to repress further GAL1-regulated ALP synthesis, and at times thereafter cells were processed for microscopy.
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fig9: Transport of ALP to the vacuole is impaired in cells with inadequate Gcs1 + Age2 activity. (A) Cells growing at 26°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 7 min, and incubated further with unlabeled amino acids. ALP was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The precursor (P) and mature (M) forms are indicated. After 30 min, wild-type cells had >90% of ALP in the mature form, whereas double-mutant cells had 75% of ALP in the precursor form. (B) Cells carrying plasmid pSN351, harboring the ALP gene PHO8 expressed from the inducible GAL1 promoter, were grown at 23°C in raffinose medium and transferred to 37°C. After a 15-min incubation, ALP synthesis was induced by the addition of galactose. After a 40-min induction period, glucose was added to repress further GAL1-regulated ALP synthesis, and at times thereafter cells were processed for microscopy.

Mentions: The transport of a type II vacuolar membrane protein, ALP, from the TGN to the vacuole proceeds by a different route than that followed by CPY. Whereas the TGN-to-vacuole pathway for CPY involves passage through the prevacuolar endosome and is dependent on clathrin (Seeger and Payne, 1992), the TGN-to-vacuole pathway for ALP bypasses the prevacuolar endosome, is clathrin-independent, and relies on the AP-3 adaptor protein complex (Cowles et al., 1997a; Stepp et al., 1997). To assess the effects of Gcs1 + Age2 activity on the transport of ALP to the vacuole we used the same pulse–chase immunoprecipitation procedure as above, with transport of newly synthesized ALP to the vacuole indicated by the accumulation of the mature form of ALP. Although both wild-type and mutant cells were equally effective at processing ALP to the mature form at a permissive temperature (unpublished data), at 37°C the gcs1-3 age2 double-mutant cells were unable to process ALP to the mature vacuolar form (Fig. 9 A). This finding suggests that Gcs1 + Age2 activity is also needed for the exit of ALP from the TGN via the ALP pathway.


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)

Transport of ALP to the vacuole is impaired in cells with inadequate Gcs1 + Age2 activity. (A) Cells growing at 26°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 7 min, and incubated further with unlabeled amino acids. ALP was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The precursor (P) and mature (M) forms are indicated. After 30 min, wild-type cells had >90% of ALP in the mature form, whereas double-mutant cells had 75% of ALP in the precursor form. (B) Cells carrying plasmid pSN351, harboring the ALP gene PHO8 expressed from the inducible GAL1 promoter, were grown at 23°C in raffinose medium and transferred to 37°C. After a 15-min incubation, ALP synthesis was induced by the addition of galactose. After a 40-min induction period, glucose was added to repress further GAL1-regulated ALP synthesis, and at times thereafter cells were processed for microscopy.
© Copyright Policy
Related In: Results  -  Collection

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

fig9: Transport of ALP to the vacuole is impaired in cells with inadequate Gcs1 + Age2 activity. (A) Cells growing at 26°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 7 min, and incubated further with unlabeled amino acids. ALP was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The precursor (P) and mature (M) forms are indicated. After 30 min, wild-type cells had >90% of ALP in the mature form, whereas double-mutant cells had 75% of ALP in the precursor form. (B) Cells carrying plasmid pSN351, harboring the ALP gene PHO8 expressed from the inducible GAL1 promoter, were grown at 23°C in raffinose medium and transferred to 37°C. After a 15-min incubation, ALP synthesis was induced by the addition of galactose. After a 40-min induction period, glucose was added to repress further GAL1-regulated ALP synthesis, and at times thereafter cells were processed for microscopy.
Mentions: The transport of a type II vacuolar membrane protein, ALP, from the TGN to the vacuole proceeds by a different route than that followed by CPY. Whereas the TGN-to-vacuole pathway for CPY involves passage through the prevacuolar endosome and is dependent on clathrin (Seeger and Payne, 1992), the TGN-to-vacuole pathway for ALP bypasses the prevacuolar endosome, is clathrin-independent, and relies on the AP-3 adaptor protein complex (Cowles et al., 1997a; Stepp et al., 1997). To assess the effects of Gcs1 + Age2 activity on the transport of ALP to the vacuole we used the same pulse–chase immunoprecipitation procedure as above, with transport of newly synthesized ALP to the vacuole indicated by the accumulation of the mature form of ALP. Although both wild-type and mutant cells were equally effective at processing ALP to the mature form at a permissive temperature (unpublished data), at 37°C the gcs1-3 age2 double-mutant cells were unable to process ALP to the mature vacuolar form (Fig. 9 A). This finding suggests that Gcs1 + Age2 activity is also needed for the exit of ALP from the TGN via the ALP pathway.

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