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Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole.

Kim J, Kamada Y, Stromhaug PE, Guan J, Hefner-Gravink A, Baba M, Scott SV, Ohsumi Y, Dunn WA, Klionsky DJ - J. Cell Biol. (2001)

Bottom Line: Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy.In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy.These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

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Cvt9 is localized to a perivacuolar compartment. (A) Localization pattern of GFPCvt9. The cvt9Δ (AHY001) strain was transformed with a plasmid encoding a GFPCvt9 fusion protein behind the CUP1 promoter (pCuGFPCVT9). Expression of GFPCvt9 was induced with 10 μM CuSO4 for 2 h, followed by labeling of the vacuoles with FM 4-64 (see Materials and Methods). Images were taken with a Leica IRM confocal microscope. GFPCvt9 mostly localizes to intense perivacuolar, punctate structures. (B and C) Subcellular localization of Cvt9 by Optiprep density gradients. The cvt9Δ strain was transformed with pCuCVT9(416) and incubated with 50 μM CuSO4 at midlog growth stage to induce Cvt9 expression. The cells were converted to spheroplasts and lysed in gradient lysis buffer (see Materials and Methods). A total membrane fraction was isolated by centrifugation at 100,000 g for 20 min and loaded to the top of a 10-ml Optiprep linear gradient (0–66%). After centrifugation at 100,000 g for 16 h at 4°C, 14 fractions were collected and analyzed by immunoblots with antiserum or antibodies to Cvt9 and (B) Pho8 (vacuole), (C) Dpm1 (ER), Kex2 (trans-Golgi Network), and Pep12 (endosome). The immunoblots and the graphs of the densitometry quantitation are from the same gradient but presented separately for clarity.
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Figure 4: Cvt9 is localized to a perivacuolar compartment. (A) Localization pattern of GFPCvt9. The cvt9Δ (AHY001) strain was transformed with a plasmid encoding a GFPCvt9 fusion protein behind the CUP1 promoter (pCuGFPCVT9). Expression of GFPCvt9 was induced with 10 μM CuSO4 for 2 h, followed by labeling of the vacuoles with FM 4-64 (see Materials and Methods). Images were taken with a Leica IRM confocal microscope. GFPCvt9 mostly localizes to intense perivacuolar, punctate structures. (B and C) Subcellular localization of Cvt9 by Optiprep density gradients. The cvt9Δ strain was transformed with pCuCVT9(416) and incubated with 50 μM CuSO4 at midlog growth stage to induce Cvt9 expression. The cells were converted to spheroplasts and lysed in gradient lysis buffer (see Materials and Methods). A total membrane fraction was isolated by centrifugation at 100,000 g for 20 min and loaded to the top of a 10-ml Optiprep linear gradient (0–66%). After centrifugation at 100,000 g for 16 h at 4°C, 14 fractions were collected and analyzed by immunoblots with antiserum or antibodies to Cvt9 and (B) Pho8 (vacuole), (C) Dpm1 (ER), Kex2 (trans-Golgi Network), and Pep12 (endosome). The immunoblots and the graphs of the densitometry quantitation are from the same gradient but presented separately for clarity.

Mentions: To investigate the identity of the membrane compartment with which Cvt9 associates, a GFP fusion to the NH2 terminus of Cvt9 was examined. Under the control of the CUP1 promoter, GFPCvt9 expression complemented the prAPI defect in the cvt9Δ strain, indicating that it was a functional chimera (data not shown). At midlog stage, cvt9Δ cells expressing GFPCvt9 were labeled with FM 4-64 to mark the vacuoles and examined by confocal microscopy. GFPCvt9 appeared to be concentrated at prominent punctate structures directly adjacent to FM 4-64–labeled vacuoles (Fig. 4 A). A weak, cytosolic distribution of GFPCvt9 was also detected, consistent with the ratio of Cvt9 found in the membrane pellet and cytosolic supernatant fractions by biochemical fractionation analysis (compare Fig. 3 B and 4 A).


Cvt9/Gsa9 functions in sequestering selective cytosolic cargo destined for the vacuole.

Kim J, Kamada Y, Stromhaug PE, Guan J, Hefner-Gravink A, Baba M, Scott SV, Ohsumi Y, Dunn WA, Klionsky DJ - J. Cell Biol. (2001)

Cvt9 is localized to a perivacuolar compartment. (A) Localization pattern of GFPCvt9. The cvt9Δ (AHY001) strain was transformed with a plasmid encoding a GFPCvt9 fusion protein behind the CUP1 promoter (pCuGFPCVT9). Expression of GFPCvt9 was induced with 10 μM CuSO4 for 2 h, followed by labeling of the vacuoles with FM 4-64 (see Materials and Methods). Images were taken with a Leica IRM confocal microscope. GFPCvt9 mostly localizes to intense perivacuolar, punctate structures. (B and C) Subcellular localization of Cvt9 by Optiprep density gradients. The cvt9Δ strain was transformed with pCuCVT9(416) and incubated with 50 μM CuSO4 at midlog growth stage to induce Cvt9 expression. The cells were converted to spheroplasts and lysed in gradient lysis buffer (see Materials and Methods). A total membrane fraction was isolated by centrifugation at 100,000 g for 20 min and loaded to the top of a 10-ml Optiprep linear gradient (0–66%). After centrifugation at 100,000 g for 16 h at 4°C, 14 fractions were collected and analyzed by immunoblots with antiserum or antibodies to Cvt9 and (B) Pho8 (vacuole), (C) Dpm1 (ER), Kex2 (trans-Golgi Network), and Pep12 (endosome). The immunoblots and the graphs of the densitometry quantitation are from the same gradient but presented separately for clarity.
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Figure 4: Cvt9 is localized to a perivacuolar compartment. (A) Localization pattern of GFPCvt9. The cvt9Δ (AHY001) strain was transformed with a plasmid encoding a GFPCvt9 fusion protein behind the CUP1 promoter (pCuGFPCVT9). Expression of GFPCvt9 was induced with 10 μM CuSO4 for 2 h, followed by labeling of the vacuoles with FM 4-64 (see Materials and Methods). Images were taken with a Leica IRM confocal microscope. GFPCvt9 mostly localizes to intense perivacuolar, punctate structures. (B and C) Subcellular localization of Cvt9 by Optiprep density gradients. The cvt9Δ strain was transformed with pCuCVT9(416) and incubated with 50 μM CuSO4 at midlog growth stage to induce Cvt9 expression. The cells were converted to spheroplasts and lysed in gradient lysis buffer (see Materials and Methods). A total membrane fraction was isolated by centrifugation at 100,000 g for 20 min and loaded to the top of a 10-ml Optiprep linear gradient (0–66%). After centrifugation at 100,000 g for 16 h at 4°C, 14 fractions were collected and analyzed by immunoblots with antiserum or antibodies to Cvt9 and (B) Pho8 (vacuole), (C) Dpm1 (ER), Kex2 (trans-Golgi Network), and Pep12 (endosome). The immunoblots and the graphs of the densitometry quantitation are from the same gradient but presented separately for clarity.
Mentions: To investigate the identity of the membrane compartment with which Cvt9 associates, a GFP fusion to the NH2 terminus of Cvt9 was examined. Under the control of the CUP1 promoter, GFPCvt9 expression complemented the prAPI defect in the cvt9Δ strain, indicating that it was a functional chimera (data not shown). At midlog stage, cvt9Δ cells expressing GFPCvt9 were labeled with FM 4-64 to mark the vacuoles and examined by confocal microscopy. GFPCvt9 appeared to be concentrated at prominent punctate structures directly adjacent to FM 4-64–labeled vacuoles (Fig. 4 A). A weak, cytosolic distribution of GFPCvt9 was also detected, consistent with the ratio of Cvt9 found in the membrane pellet and cytosolic supernatant fractions by biochemical fractionation analysis (compare Fig. 3 B and 4 A).

Bottom Line: Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy.In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy.These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Michigan, Ann Arbor, Michigan 48109, USA.

ABSTRACT
Three overlapping pathways mediate the transport of cytoplasmic material to the vacuole in Saccharomyces cerevisiae. The cytoplasm to vacuole targeting (Cvt) pathway transports the vacuolar hydrolase, aminopeptidase I (API), whereas pexophagy mediates the delivery of excess peroxisomes for degradation. Both the Cvt and pexophagy pathways are selective processes that specifically recognize their cargo. In contrast, macroautophagy nonselectively transports bulk cytosol to the vacuole for recycling. Most of the import machinery characterized thus far is required for all three modes of transport. However, unique features of each pathway dictate the requirement for additional components that differentiate these pathways from one another, including at the step of specific cargo selection.We have identified Cvt9 and its Pichia pastoris counterpart Gsa9. In S. cerevisiae, Cvt9 is required for the selective delivery of precursor API (prAPI) to the vacuole by the Cvt pathway and the targeted degradation of peroxisomes by pexophagy. In P. pastoris, Gsa9 is required for glucose-induced pexophagy. Significantly, neither Cvt9 nor Gsa9 is required for starvation-induced nonselective transport of bulk cytoplasmic cargo by macroautophagy. The deletion of CVT9 destabilizes the binding of prAPI to the membrane and analysis of a cvt9 temperature-sensitive mutant supports a direct role of Cvt9 in transport vesicle formation. Cvt9 oligomers peripherally associate with a novel, perivacuolar membrane compartment and interact with Apg1, a Ser/Thr kinase essential for both the Cvt pathway and autophagy. In P. pastoris Gsa9 is recruited to concentrated regions on the vacuole membrane that contact peroxisomes in the process of being engulfed by pexophagy. These biochemical and morphological results demonstrate that Cvt9 and the P. pastoris homologue Gsa9 may function at the step of selective cargo sequestration.

Show MeSH
Related in: MedlinePlus