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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 physically associates with Apg1. The apg1Δ strain (YYK36) was transformed with a plasmid encoding myc-tagged Cvt9 (p[myc-CVT9]) with or without an additional plasmid encoding Apg1 (p[APG1]). The cell lysates were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-Apg1 antiserum (lanes 1, 3, and 5) or buffer only (lanes 2, 4, and 6). The immunoprecipitates (IP) were analyzed by immunoblot with antiserum to myc. The anti-Apg1–precipitated immunocomplex contains myc-tagged Cvt9 (lane 5).
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Figure 5: Cvt9 physically associates with Apg1. The apg1Δ strain (YYK36) was transformed with a plasmid encoding myc-tagged Cvt9 (p[myc-CVT9]) with or without an additional plasmid encoding Apg1 (p[APG1]). The cell lysates were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-Apg1 antiserum (lanes 1, 3, and 5) or buffer only (lanes 2, 4, and 6). The immunoprecipitates (IP) were analyzed by immunoblot with antiserum to myc. The anti-Apg1–precipitated immunocomplex contains myc-tagged Cvt9 (lane 5).

Mentions: Cvt9 is present in a detergent-resistant form, presumably a protein complex, which localizes to a distinct membrane compartment (Fig. 3 and Fig. 4). We next identified an additional factor of the Cvt9 protein complex. A two-hybrid analysis to identify proteins that associate with the autophagy component Apg1 identified Cvt9 as a potential interacting partner (Kamada et al. 2000). The Apg1–Cvt9 interaction was directly supported by a coimmunoprecipitation analysis (Fig. 5). The apg1Δ strain was transformed with the plasmid encoding the myc-tagged Cvt9 fusion protein with or without a second plasmid encoding Apg1. Cell extracts were immunoprecipitated with antiserum to Apg1 under native, nondenaturing conditions as described in Materials and Methods. The precipitated immunocomplexes were then subjected to a denaturing SDS-PAGE/immunoblot procedure with antiserum to the myc epitope. In cells expressing both myc–Cvt9 and Apg1, the immunocomplexes precipitated with the Apg1 antiserum also isolated the myc-tagged Cvt9 protein, thus confirming the two-hybrid data that Apg1 and Cvt9 physically interact (Fig. 5, lane 5).


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 physically associates with Apg1. The apg1Δ strain (YYK36) was transformed with a plasmid encoding myc-tagged Cvt9 (p[myc-CVT9]) with or without an additional plasmid encoding Apg1 (p[APG1]). The cell lysates were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-Apg1 antiserum (lanes 1, 3, and 5) or buffer only (lanes 2, 4, and 6). The immunoprecipitates (IP) were analyzed by immunoblot with antiserum to myc. The anti-Apg1–precipitated immunocomplex contains myc-tagged Cvt9 (lane 5).
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Related In: Results  -  Collection

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Figure 5: Cvt9 physically associates with Apg1. The apg1Δ strain (YYK36) was transformed with a plasmid encoding myc-tagged Cvt9 (p[myc-CVT9]) with or without an additional plasmid encoding Apg1 (p[APG1]). The cell lysates were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-Apg1 antiserum (lanes 1, 3, and 5) or buffer only (lanes 2, 4, and 6). The immunoprecipitates (IP) were analyzed by immunoblot with antiserum to myc. The anti-Apg1–precipitated immunocomplex contains myc-tagged Cvt9 (lane 5).
Mentions: Cvt9 is present in a detergent-resistant form, presumably a protein complex, which localizes to a distinct membrane compartment (Fig. 3 and Fig. 4). We next identified an additional factor of the Cvt9 protein complex. A two-hybrid analysis to identify proteins that associate with the autophagy component Apg1 identified Cvt9 as a potential interacting partner (Kamada et al. 2000). The Apg1–Cvt9 interaction was directly supported by a coimmunoprecipitation analysis (Fig. 5). The apg1Δ strain was transformed with the plasmid encoding the myc-tagged Cvt9 fusion protein with or without a second plasmid encoding Apg1. Cell extracts were immunoprecipitated with antiserum to Apg1 under native, nondenaturing conditions as described in Materials and Methods. The precipitated immunocomplexes were then subjected to a denaturing SDS-PAGE/immunoblot procedure with antiserum to the myc epitope. In cells expressing both myc–Cvt9 and Apg1, the immunocomplexes precipitated with the Apg1 antiserum also isolated the myc-tagged Cvt9 protein, thus confirming the two-hybrid data that Apg1 and Cvt9 physically interact (Fig. 5, lane 5).

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