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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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GFP/HA-Gsa9 localization during glucose adaptation. ANB23 cells expressing both BFP fused at its COOH terminus to the serine-lysine-leucine (SKL) peroxisomal targeting signal (BFP-SKL) and GFP/HA-Gsa9 were grown under methanol conditions, then adapted to glucose for 4 h. The peroxisomes were labeled by BFP-SKL (middle). During glucose adaptation, the GFP/HA-Gsa9 (top) was concentrated to regions of the vacuolar membrane that engulf the peroxisomes containing the BFP-SKL marker (see overlay in bottom panels). A punctate GFP/HA-Gsa9 pattern was also detected.
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Figure 7: GFP/HA-Gsa9 localization during glucose adaptation. ANB23 cells expressing both BFP fused at its COOH terminus to the serine-lysine-leucine (SKL) peroxisomal targeting signal (BFP-SKL) and GFP/HA-Gsa9 were grown under methanol conditions, then adapted to glucose for 4 h. The peroxisomes were labeled by BFP-SKL (middle). During glucose adaptation, the GFP/HA-Gsa9 (top) was concentrated to regions of the vacuolar membrane that engulf the peroxisomes containing the BFP-SKL marker (see overlay in bottom panels). A punctate GFP/HA-Gsa9 pattern was also detected.

Mentions: To further characterize the role of Gsa9 in micropexophagy, we examined its localization by fluorescence microscopy using a functional GFP/HA-Gsa9 fusion protein. This construct was able to complement the gsa9Δ phenotype (data not shown). In cells that have not been subjected to glucose adaptation conditions, GFP/HA-Gsa9 appeared localized to one or two structures adjacent to the vacuole (data not shown). In addition, a weak GFP/HA-Gsa9 signal was detected coincident with the FM 4-64–labeled vacuoles. The punctate pattern of GFP/HA-Gsa9 appeared nearly identical to the localization pattern of GFPCvt9, although we could not detect GFPCvt9 on the vacuole (Fig. 4 A). When cells expressing GFP/HA-Gsa9 were grown in methanol medium to proliferate peroxisomes and then subjected to adaptation in glucose medium, the GFP/HA-Gsa9 signal localized predominantly to the vacuolar membrane (Fig. 7, top). To compare GFP/HA-Gsa9 localization to that of peroxisomes, we constructed a blue fluorescent protein (BFP) tagged with the serine-lysine-leucine COOH-terminal peroxisomal targeting signal (BFP-SKL; Fig. 7, middle). In glucose adaptation conditions, the intensity of GFP/HA-Gsa9 appeared strongest at the vacuolar regions that surrounded the peroxisomes during the engulfment process (Fig. 7, bottom). GFP/HA-Gsa9 also formed punctate structures that appeared continuous with its vacuolar membrane labeling pattern. The distribution of GFP/HA-Gsa9 before and after glucose adaptation suggests that Gsa9 localizes to regions of the vacuolar membrane that contact peroxisomes during micropexophagy.


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)

GFP/HA-Gsa9 localization during glucose adaptation. ANB23 cells expressing both BFP fused at its COOH terminus to the serine-lysine-leucine (SKL) peroxisomal targeting signal (BFP-SKL) and GFP/HA-Gsa9 were grown under methanol conditions, then adapted to glucose for 4 h. The peroxisomes were labeled by BFP-SKL (middle). During glucose adaptation, the GFP/HA-Gsa9 (top) was concentrated to regions of the vacuolar membrane that engulf the peroxisomes containing the BFP-SKL marker (see overlay in bottom panels). A punctate GFP/HA-Gsa9 pattern was also detected.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2169458&req=5

Figure 7: GFP/HA-Gsa9 localization during glucose adaptation. ANB23 cells expressing both BFP fused at its COOH terminus to the serine-lysine-leucine (SKL) peroxisomal targeting signal (BFP-SKL) and GFP/HA-Gsa9 were grown under methanol conditions, then adapted to glucose for 4 h. The peroxisomes were labeled by BFP-SKL (middle). During glucose adaptation, the GFP/HA-Gsa9 (top) was concentrated to regions of the vacuolar membrane that engulf the peroxisomes containing the BFP-SKL marker (see overlay in bottom panels). A punctate GFP/HA-Gsa9 pattern was also detected.
Mentions: To further characterize the role of Gsa9 in micropexophagy, we examined its localization by fluorescence microscopy using a functional GFP/HA-Gsa9 fusion protein. This construct was able to complement the gsa9Δ phenotype (data not shown). In cells that have not been subjected to glucose adaptation conditions, GFP/HA-Gsa9 appeared localized to one or two structures adjacent to the vacuole (data not shown). In addition, a weak GFP/HA-Gsa9 signal was detected coincident with the FM 4-64–labeled vacuoles. The punctate pattern of GFP/HA-Gsa9 appeared nearly identical to the localization pattern of GFPCvt9, although we could not detect GFPCvt9 on the vacuole (Fig. 4 A). When cells expressing GFP/HA-Gsa9 were grown in methanol medium to proliferate peroxisomes and then subjected to adaptation in glucose medium, the GFP/HA-Gsa9 signal localized predominantly to the vacuolar membrane (Fig. 7, top). To compare GFP/HA-Gsa9 localization to that of peroxisomes, we constructed a blue fluorescent protein (BFP) tagged with the serine-lysine-leucine COOH-terminal peroxisomal targeting signal (BFP-SKL; Fig. 7, middle). In glucose adaptation conditions, the intensity of GFP/HA-Gsa9 appeared strongest at the vacuolar regions that surrounded the peroxisomes during the engulfment process (Fig. 7, bottom). GFP/HA-Gsa9 also formed punctate structures that appeared continuous with its vacuolar membrane labeling pattern. The distribution of GFP/HA-Gsa9 before and after glucose adaptation suggests that Gsa9 localizes to regions of the vacuolar membrane that contact peroxisomes during micropexophagy.

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