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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 and the P. pastoris homologue Gsa9 are required for peroxisome degradation. (A) The S. cerevisiae cvt9Δ strain is defective in pexophagy. Peroxisomes from wild-type (WT; KA311A) and cvt9Δ (YYK107) cells were proliferated in oleic acid medium and then degraded by shifting to SD-N medium at the indicated times (see Materials and Methods). Pexophagy was monitored by immunoblot analysis of the peroxisomal thiolase enzyme, Fox3. Fox3 levels decrease in wild-type cells under pexophagy conditions but remain constant in cvt9Δ. (B) The P. pastoris gsa9 strain is defective in pexophagy. Peroxisomes from wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were proliferated in methanol medium and then degraded by shifting to glucose medium for 0 and 6 h (see Materials and Methods). Pexophagy was measured by an activity assay for the peroxisomal AOX enzyme. AOX activity decreases in wild-type cells in glucose adaptation conditions, but remains relatively high in gsa7 and gsa9 cells. (C) The P. pastoris gsa9Δ strain is not defective in autophagy. Wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were radiolabeled with 1 μCi/ml [14C]valine for 16 h. The cells were then shifted to nitrogen-depleted chase medium and the production of TCA-soluble radioactivity was measured during 2–24 h of chase. Protein turnover rate in gsa9 cells is comparable to wild-type cells but relatively low in the autophagy-defective gsa7 cells. (D) The P. pastoris gsa9 mutants are defective at a middle to late stage of micropexophagy. Wild-type strain (GS115) and mutant strains gsa9-2 (R8) and gsa9Δ (WDK09) were grown to confluency under methanol conditions to proliferate peroxisomes. The strains were then shifted to a glucose-containing medium for 2 h to induce peroxisome degradation before fixation and processing for electron microscopy (see Materials and Methods). Partially degraded peroxisomes (Px) were found within the vacuole (V) of the parental GS115 cells. This was not observed in the gsa9-2 and gsa9Δ mutants. Instead, arm-like extensions of the vacuole(s) were observed to partially surround the peroxisomes. The nuclei (N) are also indicated.
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Figure 6: Cvt9 and the P. pastoris homologue Gsa9 are required for peroxisome degradation. (A) The S. cerevisiae cvt9Δ strain is defective in pexophagy. Peroxisomes from wild-type (WT; KA311A) and cvt9Δ (YYK107) cells were proliferated in oleic acid medium and then degraded by shifting to SD-N medium at the indicated times (see Materials and Methods). Pexophagy was monitored by immunoblot analysis of the peroxisomal thiolase enzyme, Fox3. Fox3 levels decrease in wild-type cells under pexophagy conditions but remain constant in cvt9Δ. (B) The P. pastoris gsa9 strain is defective in pexophagy. Peroxisomes from wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were proliferated in methanol medium and then degraded by shifting to glucose medium for 0 and 6 h (see Materials and Methods). Pexophagy was measured by an activity assay for the peroxisomal AOX enzyme. AOX activity decreases in wild-type cells in glucose adaptation conditions, but remains relatively high in gsa7 and gsa9 cells. (C) The P. pastoris gsa9Δ strain is not defective in autophagy. Wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were radiolabeled with 1 μCi/ml [14C]valine for 16 h. The cells were then shifted to nitrogen-depleted chase medium and the production of TCA-soluble radioactivity was measured during 2–24 h of chase. Protein turnover rate in gsa9 cells is comparable to wild-type cells but relatively low in the autophagy-defective gsa7 cells. (D) The P. pastoris gsa9 mutants are defective at a middle to late stage of micropexophagy. Wild-type strain (GS115) and mutant strains gsa9-2 (R8) and gsa9Δ (WDK09) were grown to confluency under methanol conditions to proliferate peroxisomes. The strains were then shifted to a glucose-containing medium for 2 h to induce peroxisome degradation before fixation and processing for electron microscopy (see Materials and Methods). Partially degraded peroxisomes (Px) were found within the vacuole (V) of the parental GS115 cells. This was not observed in the gsa9-2 and gsa9Δ mutants. Instead, arm-like extensions of the vacuole(s) were observed to partially surround the peroxisomes. The nuclei (N) are also indicated.

Mentions: To determine if Cvt9 was also required for the specific degradation of peroxisomes in S. cerevisiae, we examined the degradation of Fox3, the peroxisomal thiolase enzyme, following a previously described method for examining pexophagy in S. cerevisiae (Hutchins et al. 1999). Peroxisome proliferation was induced in cvt9Δ and wild-type cells by growth in oleic acid medium. Upon shift to SD-N medium to induce pexophagy, peroxisome degradation was examined by monitoring cellular Fox3 levels over the indicated time course (Fig. 6 A). In wild-type cells, Fox3 levels decreased significantly over the time course, whereas they remained constant in cvt9Δ cells. This finding suggests that Cvt9 is required for the specific, vacuolar delivery of not only prAPI via the Cvt pathway, but also peroxisomes via the pexophagy pathway in S. cerevisiae.


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 and the P. pastoris homologue Gsa9 are required for peroxisome degradation. (A) The S. cerevisiae cvt9Δ strain is defective in pexophagy. Peroxisomes from wild-type (WT; KA311A) and cvt9Δ (YYK107) cells were proliferated in oleic acid medium and then degraded by shifting to SD-N medium at the indicated times (see Materials and Methods). Pexophagy was monitored by immunoblot analysis of the peroxisomal thiolase enzyme, Fox3. Fox3 levels decrease in wild-type cells under pexophagy conditions but remain constant in cvt9Δ. (B) The P. pastoris gsa9 strain is defective in pexophagy. Peroxisomes from wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were proliferated in methanol medium and then degraded by shifting to glucose medium for 0 and 6 h (see Materials and Methods). Pexophagy was measured by an activity assay for the peroxisomal AOX enzyme. AOX activity decreases in wild-type cells in glucose adaptation conditions, but remains relatively high in gsa7 and gsa9 cells. (C) The P. pastoris gsa9Δ strain is not defective in autophagy. Wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were radiolabeled with 1 μCi/ml [14C]valine for 16 h. The cells were then shifted to nitrogen-depleted chase medium and the production of TCA-soluble radioactivity was measured during 2–24 h of chase. Protein turnover rate in gsa9 cells is comparable to wild-type cells but relatively low in the autophagy-defective gsa7 cells. (D) The P. pastoris gsa9 mutants are defective at a middle to late stage of micropexophagy. Wild-type strain (GS115) and mutant strains gsa9-2 (R8) and gsa9Δ (WDK09) were grown to confluency under methanol conditions to proliferate peroxisomes. The strains were then shifted to a glucose-containing medium for 2 h to induce peroxisome degradation before fixation and processing for electron microscopy (see Materials and Methods). Partially degraded peroxisomes (Px) were found within the vacuole (V) of the parental GS115 cells. This was not observed in the gsa9-2 and gsa9Δ mutants. Instead, arm-like extensions of the vacuole(s) were observed to partially surround the peroxisomes. The nuclei (N) are also indicated.
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Figure 6: Cvt9 and the P. pastoris homologue Gsa9 are required for peroxisome degradation. (A) The S. cerevisiae cvt9Δ strain is defective in pexophagy. Peroxisomes from wild-type (WT; KA311A) and cvt9Δ (YYK107) cells were proliferated in oleic acid medium and then degraded by shifting to SD-N medium at the indicated times (see Materials and Methods). Pexophagy was monitored by immunoblot analysis of the peroxisomal thiolase enzyme, Fox3. Fox3 levels decrease in wild-type cells under pexophagy conditions but remain constant in cvt9Δ. (B) The P. pastoris gsa9 strain is defective in pexophagy. Peroxisomes from wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were proliferated in methanol medium and then degraded by shifting to glucose medium for 0 and 6 h (see Materials and Methods). Pexophagy was measured by an activity assay for the peroxisomal AOX enzyme. AOX activity decreases in wild-type cells in glucose adaptation conditions, but remains relatively high in gsa7 and gsa9 cells. (C) The P. pastoris gsa9Δ strain is not defective in autophagy. Wild-type (GS115), gsa7 (WDY7), and gsa9 (R8) cells were radiolabeled with 1 μCi/ml [14C]valine for 16 h. The cells were then shifted to nitrogen-depleted chase medium and the production of TCA-soluble radioactivity was measured during 2–24 h of chase. Protein turnover rate in gsa9 cells is comparable to wild-type cells but relatively low in the autophagy-defective gsa7 cells. (D) The P. pastoris gsa9 mutants are defective at a middle to late stage of micropexophagy. Wild-type strain (GS115) and mutant strains gsa9-2 (R8) and gsa9Δ (WDK09) were grown to confluency under methanol conditions to proliferate peroxisomes. The strains were then shifted to a glucose-containing medium for 2 h to induce peroxisome degradation before fixation and processing for electron microscopy (see Materials and Methods). Partially degraded peroxisomes (Px) were found within the vacuole (V) of the parental GS115 cells. This was not observed in the gsa9-2 and gsa9Δ mutants. Instead, arm-like extensions of the vacuole(s) were observed to partially surround the peroxisomes. The nuclei (N) are also indicated.
Mentions: To determine if Cvt9 was also required for the specific degradation of peroxisomes in S. cerevisiae, we examined the degradation of Fox3, the peroxisomal thiolase enzyme, following a previously described method for examining pexophagy in S. cerevisiae (Hutchins et al. 1999). Peroxisome proliferation was induced in cvt9Δ and wild-type cells by growth in oleic acid medium. Upon shift to SD-N medium to induce pexophagy, peroxisome degradation was examined by monitoring cellular Fox3 levels over the indicated time course (Fig. 6 A). In wild-type cells, Fox3 levels decreased significantly over the time course, whereas they remained constant in cvt9Δ cells. This finding suggests that Cvt9 is required for the specific, vacuolar delivery of not only prAPI via the Cvt pathway, but also peroxisomes via the pexophagy pathway in S. cerevisiae.

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