Limits...
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.

Show MeSH

Related in: MedlinePlus

CPY transport from the TGN is aberrant under conditions of diminished Gcs + 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. CPY was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The locations of the P1, P2, and mature (M) forms of CPY are indicated. After 30 min, wild-type cells had >90% of CPY in the mature form (lane 3), whereas the majority (68%) of CPY in double-mutant cells was in the P2 form (lane 6). For reference, lane 7 contains another 0-time sample to aid identification of the P1 and P2 forms of CPY. (B) Cells were treated as in panel A, but fractionated into external (e) and internal (i) fractions before immunoprecipitation. Although wild-type cells retained >90% of CPY internally, double-mutant cells secreted 35% of the CPY during the incubation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2199332&req=5

fig7: CPY transport from the TGN is aberrant under conditions of diminished Gcs + 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. CPY was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The locations of the P1, P2, and mature (M) forms of CPY are indicated. After 30 min, wild-type cells had >90% of CPY in the mature form (lane 3), whereas the majority (68%) of CPY in double-mutant cells was in the P2 form (lane 6). For reference, lane 7 contains another 0-time sample to aid identification of the P1 and P2 forms of CPY. (B) Cells were treated as in panel A, but fractionated into external (e) and internal (i) fractions before immunoprecipitation. Although wild-type cells retained >90% of CPY internally, double-mutant cells secreted 35% of the CPY during the incubation.

Mentions: Material transported to the vacuole includes newly synthesized vacuolar proteins that move from the ER through the Golgi, and that are sorted in the late Golgi compartments to be directed to the vacuole through the endosomal system. Therefore, a defect in vesicular transport that affects Golgi and/or endosomal function can also affect this pathway. A useful marker of this transport from the ER through the Golgi to the vacuole is a vacuolar hydrolase, CPY. In the ER, glycosylation of CPY gives rise to the precursor form p1CPY. Transit to the Golgi and extension of core oligosaccharides results in the Golgi-specific precursor p2CPY. Finally, transport of p2CPY to the vacuole via an endosomal compartment allows proteolytic maturation to yield the mature form of CPY. Impairment of transport to the vacuole can lead to defective targeting so that the vacuolar maturation step does not occur and the precursor p2CPY is exported out of the cell (Rothman and Stevens, 1986). To determine the role of Gcs1 + Age2 activity in the transport of CPY, we transferred cells to 37°C and assessed CPY processing at this temperature. Wild-type (Fig. 7 A) and single-mutant cells (unpublished data) displayed a rapid maturation of newly made CPY to the mature form, indicating normal transit of CPY to the vacuole. However, gcs1-3 age2 double-mutant cells were defective in CPY maturation (Fig. 7 A), such that the majority of newly synthesized CPY remained in the p2CPY form. This maturation blockage is consistent with impaired delivery from the TGN 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)

CPY transport from the TGN is aberrant under conditions of diminished Gcs + 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. CPY was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The locations of the P1, P2, and mature (M) forms of CPY are indicated. After 30 min, wild-type cells had >90% of CPY in the mature form (lane 3), whereas the majority (68%) of CPY in double-mutant cells was in the P2 form (lane 6). For reference, lane 7 contains another 0-time sample to aid identification of the P1 and P2 forms of CPY. (B) Cells were treated as in panel A, but fractionated into external (e) and internal (i) fractions before immunoprecipitation. Although wild-type cells retained >90% of CPY internally, double-mutant cells secreted 35% of the CPY during the incubation.
© Copyright Policy
Related In: Results  -  Collection

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

fig7: CPY transport from the TGN is aberrant under conditions of diminished Gcs + 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. CPY was immunoprecipitated from samples removed at the indicated times, resolved by SDS-PAGE, and detected by autoradiography. The locations of the P1, P2, and mature (M) forms of CPY are indicated. After 30 min, wild-type cells had >90% of CPY in the mature form (lane 3), whereas the majority (68%) of CPY in double-mutant cells was in the P2 form (lane 6). For reference, lane 7 contains another 0-time sample to aid identification of the P1 and P2 forms of CPY. (B) Cells were treated as in panel A, but fractionated into external (e) and internal (i) fractions before immunoprecipitation. Although wild-type cells retained >90% of CPY internally, double-mutant cells secreted 35% of the CPY during the incubation.
Mentions: Material transported to the vacuole includes newly synthesized vacuolar proteins that move from the ER through the Golgi, and that are sorted in the late Golgi compartments to be directed to the vacuole through the endosomal system. Therefore, a defect in vesicular transport that affects Golgi and/or endosomal function can also affect this pathway. A useful marker of this transport from the ER through the Golgi to the vacuole is a vacuolar hydrolase, CPY. In the ER, glycosylation of CPY gives rise to the precursor form p1CPY. Transit to the Golgi and extension of core oligosaccharides results in the Golgi-specific precursor p2CPY. Finally, transport of p2CPY to the vacuole via an endosomal compartment allows proteolytic maturation to yield the mature form of CPY. Impairment of transport to the vacuole can lead to defective targeting so that the vacuolar maturation step does not occur and the precursor p2CPY is exported out of the cell (Rothman and Stevens, 1986). To determine the role of Gcs1 + Age2 activity in the transport of CPY, we transferred cells to 37°C and assessed CPY processing at this temperature. Wild-type (Fig. 7 A) and single-mutant cells (unpublished data) displayed a rapid maturation of newly made CPY to the mature form, indicating normal transit of CPY to the vacuole. However, gcs1-3 age2 double-mutant cells were defective in CPY maturation (Fig. 7 A), such that the majority of newly synthesized CPY remained in the p2CPY form. This maturation blockage is consistent with impaired delivery from the TGN 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