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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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Characterization of Cvt9. (A) Cvt9 forms a homodimer. The wild-type (KA311A) strain was transformed with a plasmid encoding myc-tagged Cvt9 with or without a second plasmid for HA-tagged Cvt9. Cell lysates were prepared as described in Materials and Methods and immunoprecipitated (IP) with antiserum to HA (lanes 1 and 3) or buffer control (lanes 2 and 4). The immunoprecipitates were then immunoblotted with antiserum or antibodies to HA and myc. The anti-HA–precipitated immunocomplex also contains myc-tagged Cvt9. The cell lysates were also directly immunoblotted with anti-HA and anti-myc antibodies to assess the expression of the epitope tagged Cvt9 fusions (lanes 5 and 6). (B) Cvt9 subcellular fractionation pattern. The cvt9Δ (AHY001) strain was transformed with a CVT9 plasmid behind the CUP1 copper-inducible promoter (pCuCVT9[416]). Expression was induced with 50 μM CuSO4 for 1 h before spheroplasting. The spheroplasts were osmotically lysed (see Materials and Methods). After a preclearing centrifugation step at 100 g for 5 min to remove unlysed spheroplasts, the total lysate (T) was separated into low speed supernatant (S13) and pellet (P13) fractions. The S13 fraction was further separated into 100,000 g supernatant (S100) and pellet (P100) fractions. The fractionated samples were subjected to immunoblot analysis using antiserum to Cvt9 and the cytosolic marker protein PGK. (C) Cvt9 is a peripheral membrane protein that is detergent resistant. A total lysate was prepared as in B and resuspended in osmotic lysis buffer only or containing 1% Triton X-100, 6 M urea, 0.1 M Na2CO3, pH 10.5, or a 1.0 M salt wash (0.67 M KOAc, 0.3 M KCl). The treated lysates were separated into total membrane (P) and supernatant (S) fractions by centrifugation at 100,000 g for 20 min at 4°C. The samples were analyzed by immunoblots with antiserum to Cvt9 and antibodies to the ER integral membrane protein Dpm1.
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Figure 3: Characterization of Cvt9. (A) Cvt9 forms a homodimer. The wild-type (KA311A) strain was transformed with a plasmid encoding myc-tagged Cvt9 with or without a second plasmid for HA-tagged Cvt9. Cell lysates were prepared as described in Materials and Methods and immunoprecipitated (IP) with antiserum to HA (lanes 1 and 3) or buffer control (lanes 2 and 4). The immunoprecipitates were then immunoblotted with antiserum or antibodies to HA and myc. The anti-HA–precipitated immunocomplex also contains myc-tagged Cvt9. The cell lysates were also directly immunoblotted with anti-HA and anti-myc antibodies to assess the expression of the epitope tagged Cvt9 fusions (lanes 5 and 6). (B) Cvt9 subcellular fractionation pattern. The cvt9Δ (AHY001) strain was transformed with a CVT9 plasmid behind the CUP1 copper-inducible promoter (pCuCVT9[416]). Expression was induced with 50 μM CuSO4 for 1 h before spheroplasting. The spheroplasts were osmotically lysed (see Materials and Methods). After a preclearing centrifugation step at 100 g for 5 min to remove unlysed spheroplasts, the total lysate (T) was separated into low speed supernatant (S13) and pellet (P13) fractions. The S13 fraction was further separated into 100,000 g supernatant (S100) and pellet (P100) fractions. The fractionated samples were subjected to immunoblot analysis using antiserum to Cvt9 and the cytosolic marker protein PGK. (C) Cvt9 is a peripheral membrane protein that is detergent resistant. A total lysate was prepared as in B and resuspended in osmotic lysis buffer only or containing 1% Triton X-100, 6 M urea, 0.1 M Na2CO3, pH 10.5, or a 1.0 M salt wash (0.67 M KOAc, 0.3 M KCl). The treated lysates were separated into total membrane (P) and supernatant (S) fractions by centrifugation at 100,000 g for 20 min at 4°C. The samples were analyzed by immunoblots with antiserum to Cvt9 and antibodies to the ER integral membrane protein Dpm1.

Mentions: To further understand Cvt9 function in the Cvt pathway, we next characterized the biosynthesis of the Cvt9 protein. Polyclonal antiserum raised against synthetic Cvt9 peptides recognized a 135-kD band by immunoblot analysis, in agreement with its predicted molecular mass (data not shown). Detection of Cvt9 increased in cvt9Δ cells transformed with the CVT9 multicopy plasmid and was absent in the cvt9Δ cells, suggesting that the antiserum is specific for the Cvt9 protein. Analysis of the Cvt9 amino acid sequence reveals a significant coiled coil secondary structure between amino acid residues 542 and 851 (http://nightingale.lcs.mit.edu/cgi-bin/score). The P. pastoris homologue, Gsa9, also contains a predicted coiled coil secondary structure motif between amino acids 811 and 1027. Because a common feature of proteins containing a coiled coil domain is the ability to form higher-ordered quaternary structures, we investigated the possibility that Cvt9 forms oligomers. Extracts from cells expressing NH2-terminally myc-tagged Cvt9 and HA-tagged Cvt9 were prepared and the immunocomplex containing HA-Cvt9 was immunoprecipitated under native, nonreducing conditions with antiserum to the HA epitope. A subsequent anti-myc epitope immunoblot detected the myc-tagged Cvt9 from the original immunoprecipitated HA–Cvt9 immunocomplex, indicating that myc-Cvt9 associates with HA–Cvt9 (Fig. 3 A). These findings indicate that Cvt9 forms homodimers or potentially larger oligomeric structures.


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)

Characterization of Cvt9. (A) Cvt9 forms a homodimer. The wild-type (KA311A) strain was transformed with a plasmid encoding myc-tagged Cvt9 with or without a second plasmid for HA-tagged Cvt9. Cell lysates were prepared as described in Materials and Methods and immunoprecipitated (IP) with antiserum to HA (lanes 1 and 3) or buffer control (lanes 2 and 4). The immunoprecipitates were then immunoblotted with antiserum or antibodies to HA and myc. The anti-HA–precipitated immunocomplex also contains myc-tagged Cvt9. The cell lysates were also directly immunoblotted with anti-HA and anti-myc antibodies to assess the expression of the epitope tagged Cvt9 fusions (lanes 5 and 6). (B) Cvt9 subcellular fractionation pattern. The cvt9Δ (AHY001) strain was transformed with a CVT9 plasmid behind the CUP1 copper-inducible promoter (pCuCVT9[416]). Expression was induced with 50 μM CuSO4 for 1 h before spheroplasting. The spheroplasts were osmotically lysed (see Materials and Methods). After a preclearing centrifugation step at 100 g for 5 min to remove unlysed spheroplasts, the total lysate (T) was separated into low speed supernatant (S13) and pellet (P13) fractions. The S13 fraction was further separated into 100,000 g supernatant (S100) and pellet (P100) fractions. The fractionated samples were subjected to immunoblot analysis using antiserum to Cvt9 and the cytosolic marker protein PGK. (C) Cvt9 is a peripheral membrane protein that is detergent resistant. A total lysate was prepared as in B and resuspended in osmotic lysis buffer only or containing 1% Triton X-100, 6 M urea, 0.1 M Na2CO3, pH 10.5, or a 1.0 M salt wash (0.67 M KOAc, 0.3 M KCl). The treated lysates were separated into total membrane (P) and supernatant (S) fractions by centrifugation at 100,000 g for 20 min at 4°C. The samples were analyzed by immunoblots with antiserum to Cvt9 and antibodies to the ER integral membrane protein Dpm1.
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Related In: Results  -  Collection

Show All Figures
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Figure 3: Characterization of Cvt9. (A) Cvt9 forms a homodimer. The wild-type (KA311A) strain was transformed with a plasmid encoding myc-tagged Cvt9 with or without a second plasmid for HA-tagged Cvt9. Cell lysates were prepared as described in Materials and Methods and immunoprecipitated (IP) with antiserum to HA (lanes 1 and 3) or buffer control (lanes 2 and 4). The immunoprecipitates were then immunoblotted with antiserum or antibodies to HA and myc. The anti-HA–precipitated immunocomplex also contains myc-tagged Cvt9. The cell lysates were also directly immunoblotted with anti-HA and anti-myc antibodies to assess the expression of the epitope tagged Cvt9 fusions (lanes 5 and 6). (B) Cvt9 subcellular fractionation pattern. The cvt9Δ (AHY001) strain was transformed with a CVT9 plasmid behind the CUP1 copper-inducible promoter (pCuCVT9[416]). Expression was induced with 50 μM CuSO4 for 1 h before spheroplasting. The spheroplasts were osmotically lysed (see Materials and Methods). After a preclearing centrifugation step at 100 g for 5 min to remove unlysed spheroplasts, the total lysate (T) was separated into low speed supernatant (S13) and pellet (P13) fractions. The S13 fraction was further separated into 100,000 g supernatant (S100) and pellet (P100) fractions. The fractionated samples were subjected to immunoblot analysis using antiserum to Cvt9 and the cytosolic marker protein PGK. (C) Cvt9 is a peripheral membrane protein that is detergent resistant. A total lysate was prepared as in B and resuspended in osmotic lysis buffer only or containing 1% Triton X-100, 6 M urea, 0.1 M Na2CO3, pH 10.5, or a 1.0 M salt wash (0.67 M KOAc, 0.3 M KCl). The treated lysates were separated into total membrane (P) and supernatant (S) fractions by centrifugation at 100,000 g for 20 min at 4°C. The samples were analyzed by immunoblots with antiserum to Cvt9 and antibodies to the ER integral membrane protein Dpm1.
Mentions: To further understand Cvt9 function in the Cvt pathway, we next characterized the biosynthesis of the Cvt9 protein. Polyclonal antiserum raised against synthetic Cvt9 peptides recognized a 135-kD band by immunoblot analysis, in agreement with its predicted molecular mass (data not shown). Detection of Cvt9 increased in cvt9Δ cells transformed with the CVT9 multicopy plasmid and was absent in the cvt9Δ cells, suggesting that the antiserum is specific for the Cvt9 protein. Analysis of the Cvt9 amino acid sequence reveals a significant coiled coil secondary structure between amino acid residues 542 and 851 (http://nightingale.lcs.mit.edu/cgi-bin/score). The P. pastoris homologue, Gsa9, also contains a predicted coiled coil secondary structure motif between amino acids 811 and 1027. Because a common feature of proteins containing a coiled coil domain is the ability to form higher-ordered quaternary structures, we investigated the possibility that Cvt9 forms oligomers. Extracts from cells expressing NH2-terminally myc-tagged Cvt9 and HA-tagged Cvt9 were prepared and the immunocomplex containing HA-Cvt9 was immunoprecipitated under native, nonreducing conditions with antiserum to the HA epitope. A subsequent anti-myc epitope immunoblot detected the myc-tagged Cvt9 from the original immunoprecipitated HA–Cvt9 immunocomplex, indicating that myc-Cvt9 associates with HA–Cvt9 (Fig. 3 A). These findings indicate that Cvt9 forms homodimers or potentially larger oligomeric structures.

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