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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 mutations destabilize prAPI membrane binding and cause a defect in vesicle formation. (A) prAPI membrane binding is destabilized in the cvt9Δ mutant. The cvt9Δ (AHY001) and apg9Δ (JKY007) strains were grown to midlog phase and converted to spheroplasts. The spheroplasts were lysed in osmotic lysis buffer supplemented with 0, 1, 2, or 5 mM MgCl2. The total lysate (T) was separated into low speed supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. The fractionated samples were subjected to immunoblot analysis using antiserum to API. (B) The temperature-conditional cvt9td strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (WT; SEY6210) and cvt9td (JGY9td) strains were incubated at 30°C and 37°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API as described in Materials and Methods. (C) prAPI in the cvt9td strain is protease accessible. Spheroplasts isolated from cvt9td and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 37°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 2,300-g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. (D) prAPI in the cvt9td strain associates with a floatable membrane fraction. Spheroplasts of apg9ts and cvt9td were osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 and added to the bottom of a step gradient of 13 and 2% Ficoll-400. The step gradients were centrifuged at 16,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods.
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Figure 2: CVT9 mutations destabilize prAPI membrane binding and cause a defect in vesicle formation. (A) prAPI membrane binding is destabilized in the cvt9Δ mutant. The cvt9Δ (AHY001) and apg9Δ (JKY007) strains were grown to midlog phase and converted to spheroplasts. The spheroplasts were lysed in osmotic lysis buffer supplemented with 0, 1, 2, or 5 mM MgCl2. The total lysate (T) was separated into low speed supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. The fractionated samples were subjected to immunoblot analysis using antiserum to API. (B) The temperature-conditional cvt9td strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (WT; SEY6210) and cvt9td (JGY9td) strains were incubated at 30°C and 37°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API as described in Materials and Methods. (C) prAPI in the cvt9td strain is protease accessible. Spheroplasts isolated from cvt9td and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 37°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 2,300-g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. (D) prAPI in the cvt9td strain associates with a floatable membrane fraction. Spheroplasts of apg9ts and cvt9td were osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 and added to the bottom of a step gradient of 13 and 2% Ficoll-400. The step gradients were centrifuged at 16,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods.

Mentions: The delivery of prAPI via the Cvt pathway involves the assembly of prAPI oligomers into a Cvt complex that binds a pelletable membrane fraction in a salt-dependent manner (Kim et al. 1997). Subcellular fractionation experiments indicate that prAPI can be recovered entirely in a low-speed pellet fraction in an osmotic lysis buffer containing 5 mM MgCl2, whereas omitting MgCl2 from the lysis buffer results in a completely cytosolic distribution of prAPI (Oda et al. 1996). Furthermore, in vitro reconstitution experiments in buffer containing 5 mM MgCl2 demonstrate that membrane-bound prAPI can subsequently be transported into the vacuole, suggesting that the salt-dependent membrane binding of prAPI represents a bona fide step in the transport pathway and not a byproduct of protein aggregation (Scott et al. 1996). We next examined the function of Cvt9 in the context of the stability of prAPI binding to the membrane fraction. The cvt9Δ cells were grown to midlog phase and converted into spheroplasts. The spheroplasts were lysed in an osmotic lysis buffer in the presence of titrating concentrations of MgCl2. The lysates were then separated into low-speed supernatant and pellet fractions and analyzed by immunoblots using antiserum to API as described in Materials and Methods. The apg9Δ strain was used as a control in which prAPI displays typical membrane-binding properties. In the absence of salt, prAPI association with the pellet fraction is severely perturbed (Fig. 2 A), consistent with previous reports (Oda et al. 1996). In a typical cvt/apg mutant such as apg9Δ, addition of increasing concentrations of MgCl2 stabilizes the binding of prAPI to the membrane fraction; substantial prAPI binding occurred at 1 mM MgCl2 and nearly complete prAPI association with the pellet fraction was detected in 5 mM MgCl2. In contrast, in the cvt9Δ strain, prAPI remained largely in the supernatant fraction even at 2 mM MgCl2, with significant prAPI membrane binding occurring only at 5 mM MgCl2. These results indicate that although Cvt9 is not essential for prAPI membrane binding, the deletion of CVT9 destabilizes the interactions between prAPI oligomers and the membrane structure to which they bind.


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 mutations destabilize prAPI membrane binding and cause a defect in vesicle formation. (A) prAPI membrane binding is destabilized in the cvt9Δ mutant. The cvt9Δ (AHY001) and apg9Δ (JKY007) strains were grown to midlog phase and converted to spheroplasts. The spheroplasts were lysed in osmotic lysis buffer supplemented with 0, 1, 2, or 5 mM MgCl2. The total lysate (T) was separated into low speed supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. The fractionated samples were subjected to immunoblot analysis using antiserum to API. (B) The temperature-conditional cvt9td strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (WT; SEY6210) and cvt9td (JGY9td) strains were incubated at 30°C and 37°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API as described in Materials and Methods. (C) prAPI in the cvt9td strain is protease accessible. Spheroplasts isolated from cvt9td and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 37°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 2,300-g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. (D) prAPI in the cvt9td strain associates with a floatable membrane fraction. Spheroplasts of apg9ts and cvt9td were osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 and added to the bottom of a step gradient of 13 and 2% Ficoll-400. The step gradients were centrifuged at 16,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods.
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Related In: Results  -  Collection

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Figure 2: CVT9 mutations destabilize prAPI membrane binding and cause a defect in vesicle formation. (A) prAPI membrane binding is destabilized in the cvt9Δ mutant. The cvt9Δ (AHY001) and apg9Δ (JKY007) strains were grown to midlog phase and converted to spheroplasts. The spheroplasts were lysed in osmotic lysis buffer supplemented with 0, 1, 2, or 5 mM MgCl2. The total lysate (T) was separated into low speed supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. The fractionated samples were subjected to immunoblot analysis using antiserum to API. (B) The temperature-conditional cvt9td strain is tightly blocked for prAPI import at nonpermissive temperature. Wild-type (WT; SEY6210) and cvt9td (JGY9td) strains were incubated at 30°C and 37°C for 5 min, pulse-labeled for 10 min, and then subjected to nonradioactive chase reactions for the indicated times. Samples at each time point were immunoprecipitated with antiserum to API as described in Materials and Methods. (C) prAPI in the cvt9td strain is protease accessible. Spheroplasts isolated from cvt9td and ypt7Δ cells were pulse-labeled for 10 min and chased for 30 min at 37°C. The labeled spheroplasts were then osmotically lysed and separated into low-speed supernatant (S) and pellet (P) fractions after a 2,300-g centrifugation step. The pellet fractions were subjected to protease treatment in the absence or presence of 0.2% Triton X-100 as described in Materials and Methods. (D) prAPI in the cvt9td strain associates with a floatable membrane fraction. Spheroplasts of apg9ts and cvt9td were osmotically lysed and separated into supernatant (S) and pellet (P) fractions by centrifugation at 2,300 g for 5 min. An aliquot was removed for the total lysate control (T). The pellet fraction (P) was resuspended in 15% Ficoll-400 and added to the bottom of a step gradient of 13 and 2% Ficoll-400. The step gradients were centrifuged at 16,000 g for 10 min. Membrane-containing float (F), nonfloat (NF), and pellet (P2) fractions were immunoprecipitated with antiserum to API as described in Materials and Methods.
Mentions: The delivery of prAPI via the Cvt pathway involves the assembly of prAPI oligomers into a Cvt complex that binds a pelletable membrane fraction in a salt-dependent manner (Kim et al. 1997). Subcellular fractionation experiments indicate that prAPI can be recovered entirely in a low-speed pellet fraction in an osmotic lysis buffer containing 5 mM MgCl2, whereas omitting MgCl2 from the lysis buffer results in a completely cytosolic distribution of prAPI (Oda et al. 1996). Furthermore, in vitro reconstitution experiments in buffer containing 5 mM MgCl2 demonstrate that membrane-bound prAPI can subsequently be transported into the vacuole, suggesting that the salt-dependent membrane binding of prAPI represents a bona fide step in the transport pathway and not a byproduct of protein aggregation (Scott et al. 1996). We next examined the function of Cvt9 in the context of the stability of prAPI binding to the membrane fraction. The cvt9Δ cells were grown to midlog phase and converted into spheroplasts. The spheroplasts were lysed in an osmotic lysis buffer in the presence of titrating concentrations of MgCl2. The lysates were then separated into low-speed supernatant and pellet fractions and analyzed by immunoblots using antiserum to API as described in Materials and Methods. The apg9Δ strain was used as a control in which prAPI displays typical membrane-binding properties. In the absence of salt, prAPI association with the pellet fraction is severely perturbed (Fig. 2 A), consistent with previous reports (Oda et al. 1996). In a typical cvt/apg mutant such as apg9Δ, addition of increasing concentrations of MgCl2 stabilizes the binding of prAPI to the membrane fraction; substantial prAPI binding occurred at 1 mM MgCl2 and nearly complete prAPI association with the pellet fraction was detected in 5 mM MgCl2. In contrast, in the cvt9Δ strain, prAPI remained largely in the supernatant fraction even at 2 mM MgCl2, with significant prAPI membrane binding occurring only at 5 mM MgCl2. These results indicate that although Cvt9 is not essential for prAPI membrane binding, the deletion of CVT9 destabilizes the interactions between prAPI oligomers and the membrane structure to which they bind.

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