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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 Gcs + Age2 proteins mediate normal trafficking of the CPY receptor Vps10. (A) Cells growing at 23°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 10 min, and incubated further with unlabeled amino acids. Vps10 was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE and detected by autoradiography. (B) Cells carrying the VPS10::3xHA allele growing at 23°C were incubated at 37°C for 2 h, fixed, stained with both anti-HA and anti-Vma2 antibodies, and visualized by DIC optics and fluorescence microscopy. (C) Wild-type and double-mutant cells growing at 23°C were incubated at 37°C for 2 h, fixed, stained with anti-Kex2 antibodies, and visualized by fluorescence microscopy.
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fig8: The Gcs + Age2 proteins mediate normal trafficking of the CPY receptor Vps10. (A) Cells growing at 23°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 10 min, and incubated further with unlabeled amino acids. Vps10 was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE and detected by autoradiography. (B) Cells carrying the VPS10::3xHA allele growing at 23°C were incubated at 37°C for 2 h, fixed, stained with both anti-HA and anti-Vma2 antibodies, and visualized by DIC optics and fluorescence microscopy. (C) Wild-type and double-mutant cells growing at 23°C were incubated at 37°C for 2 h, fixed, stained with anti-Kex2 antibodies, and visualized by fluorescence microscopy.

Mentions: Entry of CPY into a TGN-to-vacuole pathway is facilitated by a sorting receptor, Vps10, that binds CPY at the TGN (Marcusson et al., 1994; Cooper and Stevens, 1996). Transport vesicles deliver the Vps10–CPY complex to a late endosomal compartment, the prevacuolar compartment (PVC), where Vps10 is thought to uncouple from CPY. Vps10 is then packaged into vesicles for retrieval to the TGN in a manner dependent on the retromer complex (Seaman et al., 1998). In cells mutated for the PVC retrieval apparatus, Vps10 is mislocalized to the vacuole, where it is degraded (Nothwehr and Hindes, 1997; Seaman et al., 1997). Therefore, the transport block for p2CPY in gcs1-3 age2 double-mutant cells may reflect altered transport or defective localization of Vps10. To test these ideas, cells were transferred to 37°C and Vps10 stability was assessed by a pulse–chase immunoprecipitation procedure (Fig. 8 A). In gcs1-3 age2 double-mutant cells, the stability of Vps10 was indistinguishable from that in wild-type or single-mutant cells, suggesting that Vps10 is not mislocalized to the vacuole.


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 Gcs + Age2 proteins mediate normal trafficking of the CPY receptor Vps10. (A) Cells growing at 23°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 10 min, and incubated further with unlabeled amino acids. Vps10 was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE and detected by autoradiography. (B) Cells carrying the VPS10::3xHA allele growing at 23°C were incubated at 37°C for 2 h, fixed, stained with both anti-HA and anti-Vma2 antibodies, and visualized by DIC optics and fluorescence microscopy. (C) Wild-type and double-mutant cells growing at 23°C were incubated at 37°C for 2 h, fixed, stained with anti-Kex2 antibodies, and visualized by fluorescence microscopy.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199332&req=5

fig8: The Gcs + Age2 proteins mediate normal trafficking of the CPY receptor Vps10. (A) Cells growing at 23°C were incubated at 37°C for 15 min, exposed to radiolabeled amino acids for 10 min, and incubated further with unlabeled amino acids. Vps10 was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE and detected by autoradiography. (B) Cells carrying the VPS10::3xHA allele growing at 23°C were incubated at 37°C for 2 h, fixed, stained with both anti-HA and anti-Vma2 antibodies, and visualized by DIC optics and fluorescence microscopy. (C) Wild-type and double-mutant cells growing at 23°C were incubated at 37°C for 2 h, fixed, stained with anti-Kex2 antibodies, and visualized by fluorescence microscopy.
Mentions: Entry of CPY into a TGN-to-vacuole pathway is facilitated by a sorting receptor, Vps10, that binds CPY at the TGN (Marcusson et al., 1994; Cooper and Stevens, 1996). Transport vesicles deliver the Vps10–CPY complex to a late endosomal compartment, the prevacuolar compartment (PVC), where Vps10 is thought to uncouple from CPY. Vps10 is then packaged into vesicles for retrieval to the TGN in a manner dependent on the retromer complex (Seaman et al., 1998). In cells mutated for the PVC retrieval apparatus, Vps10 is mislocalized to the vacuole, where it is degraded (Nothwehr and Hindes, 1997; Seaman et al., 1997). Therefore, the transport block for p2CPY in gcs1-3 age2 double-mutant cells may reflect altered transport or defective localization of Vps10. To test these ideas, cells were transferred to 37°C and Vps10 stability was assessed by a pulse–chase immunoprecipitation procedure (Fig. 8 A). In gcs1-3 age2 double-mutant cells, the stability of Vps10 was indistinguishable from that in wild-type or single-mutant cells, suggesting that Vps10 is not mislocalized to the vacuole.

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