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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 is essential for the Cvt pathway but not for autophagy. (A) CVT9 complements the prAPI transport defect in the cvt9 mutants. Wild-type (WT; SEY6210), cvt9 (AHY96), and cvt9Δ (AHY001) strains transformed with either the centromeric (CEN) or multicopy (2μ) CVT9 plasmid (pCVT9) were grown to midlog phase in SMD. Protein extracts were prepared and analyzed by immunoblots using antiserum to API. The positions of precursor and mature API are indicated. (B) The cvt9Δ strain is largely resistant to nitrogen starvation conditions. Wild-type (SEY6210), cvt9Δ (AHY001), and cvt10/apg1 (AHY1468) cells were grown to midlog phase in SMD and transferred to SD-N as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after 2–3 d. (C) The accumulation of prAPI in cvt9Δ can be partially reversed by nitrogen starvation. The apg7Δ (VDY101), cvt3 (THY119), and cvt9Δ (AHY001) strains were grown to midlog phase and shifted to SD-N. Protein extracts were prepared at the indicated times and analyzed by immunoblots with antiserum to API. (D) ALP activity assay for autophagy. Wild-type (TN125), apg1Δ (YYK126), and cvt9Δ (YYK127) cells expressing Pho8Δ60 were shifted from nutrient-rich medium (white bars) to SD-N nitrogen-depleted medium (black bars) for 4 h. The level of autophagy induction was determined by an ALP activity assay as described in Materials and Methods. The graph is the average of three experiments. The cvt9Δ strain is not defective in the vacuolar delivery of the autophagy marker Pho8Δ60 during starvation conditions. (E) Cvt9 is not required for the formation of autophagosomes. The cvt9Δ (YYK127) strain was grown in nutrient-rich (YPD) and nitrogen starvation (SD-N) conditions supplemented with the protease inhibitor PMSF and examined by electron microscopy as described in Materials and Methods. In YPD, Cvt complexes (indicated by an arrow) could be detected in the cytoplasm. In SD-N, cvt9Δ cells accumulated autophagic bodies in the vacuole when treated with PMSF.
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Figure 1: Cvt9 is essential for the Cvt pathway but not for autophagy. (A) CVT9 complements the prAPI transport defect in the cvt9 mutants. Wild-type (WT; SEY6210), cvt9 (AHY96), and cvt9Δ (AHY001) strains transformed with either the centromeric (CEN) or multicopy (2μ) CVT9 plasmid (pCVT9) were grown to midlog phase in SMD. Protein extracts were prepared and analyzed by immunoblots using antiserum to API. The positions of precursor and mature API are indicated. (B) The cvt9Δ strain is largely resistant to nitrogen starvation conditions. Wild-type (SEY6210), cvt9Δ (AHY001), and cvt10/apg1 (AHY1468) cells were grown to midlog phase in SMD and transferred to SD-N as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after 2–3 d. (C) The accumulation of prAPI in cvt9Δ can be partially reversed by nitrogen starvation. The apg7Δ (VDY101), cvt3 (THY119), and cvt9Δ (AHY001) strains were grown to midlog phase and shifted to SD-N. Protein extracts were prepared at the indicated times and analyzed by immunoblots with antiserum to API. (D) ALP activity assay for autophagy. Wild-type (TN125), apg1Δ (YYK126), and cvt9Δ (YYK127) cells expressing Pho8Δ60 were shifted from nutrient-rich medium (white bars) to SD-N nitrogen-depleted medium (black bars) for 4 h. The level of autophagy induction was determined by an ALP activity assay as described in Materials and Methods. The graph is the average of three experiments. The cvt9Δ strain is not defective in the vacuolar delivery of the autophagy marker Pho8Δ60 during starvation conditions. (E) Cvt9 is not required for the formation of autophagosomes. The cvt9Δ (YYK127) strain was grown in nutrient-rich (YPD) and nitrogen starvation (SD-N) conditions supplemented with the protease inhibitor PMSF and examined by electron microscopy as described in Materials and Methods. In YPD, Cvt complexes (indicated by an arrow) could be detected in the cytoplasm. In SD-N, cvt9Δ cells accumulated autophagic bodies in the vacuole when treated with PMSF.

Mentions: The CVT9 single-copy plasmid rescued the prAPI processing defect in the cvt9 mutant strain (Fig. 1 A), although overexpression of Cvt9 using a multicopy CVT9 plasmid resulted in a moderate accumulation of prAPI. Although the CVT9 clone was isolated using the nitrogen starvation strategy, the viability of the cvt9- strain remained relatively robust in starvation conditions when compared with a typical mutant in the autophagy pathway (e.g., cvt10/apg1; Scott et al. 1996; Fig. 1 B). Viability under starvation conditions reflects a capacity for autophagy, indicating an ability to recycle cytosolic material after delivery to the vacuole. Therefore, we next determined if the resistance to nitrogen starvation exhibited by the cvt9Δ strain reflects its ability to carry out autophagy.


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 is essential for the Cvt pathway but not for autophagy. (A) CVT9 complements the prAPI transport defect in the cvt9 mutants. Wild-type (WT; SEY6210), cvt9 (AHY96), and cvt9Δ (AHY001) strains transformed with either the centromeric (CEN) or multicopy (2μ) CVT9 plasmid (pCVT9) were grown to midlog phase in SMD. Protein extracts were prepared and analyzed by immunoblots using antiserum to API. The positions of precursor and mature API are indicated. (B) The cvt9Δ strain is largely resistant to nitrogen starvation conditions. Wild-type (SEY6210), cvt9Δ (AHY001), and cvt10/apg1 (AHY1468) cells were grown to midlog phase in SMD and transferred to SD-N as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after 2–3 d. (C) The accumulation of prAPI in cvt9Δ can be partially reversed by nitrogen starvation. The apg7Δ (VDY101), cvt3 (THY119), and cvt9Δ (AHY001) strains were grown to midlog phase and shifted to SD-N. Protein extracts were prepared at the indicated times and analyzed by immunoblots with antiserum to API. (D) ALP activity assay for autophagy. Wild-type (TN125), apg1Δ (YYK126), and cvt9Δ (YYK127) cells expressing Pho8Δ60 were shifted from nutrient-rich medium (white bars) to SD-N nitrogen-depleted medium (black bars) for 4 h. The level of autophagy induction was determined by an ALP activity assay as described in Materials and Methods. The graph is the average of three experiments. The cvt9Δ strain is not defective in the vacuolar delivery of the autophagy marker Pho8Δ60 during starvation conditions. (E) Cvt9 is not required for the formation of autophagosomes. The cvt9Δ (YYK127) strain was grown in nutrient-rich (YPD) and nitrogen starvation (SD-N) conditions supplemented with the protease inhibitor PMSF and examined by electron microscopy as described in Materials and Methods. In YPD, Cvt complexes (indicated by an arrow) could be detected in the cytoplasm. In SD-N, cvt9Δ cells accumulated autophagic bodies in the vacuole when treated with PMSF.
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Related In: Results  -  Collection

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Figure 1: Cvt9 is essential for the Cvt pathway but not for autophagy. (A) CVT9 complements the prAPI transport defect in the cvt9 mutants. Wild-type (WT; SEY6210), cvt9 (AHY96), and cvt9Δ (AHY001) strains transformed with either the centromeric (CEN) or multicopy (2μ) CVT9 plasmid (pCVT9) were grown to midlog phase in SMD. Protein extracts were prepared and analyzed by immunoblots using antiserum to API. The positions of precursor and mature API are indicated. (B) The cvt9Δ strain is largely resistant to nitrogen starvation conditions. Wild-type (SEY6210), cvt9Δ (AHY001), and cvt10/apg1 (AHY1468) cells were grown to midlog phase in SMD and transferred to SD-N as described in Materials and Methods. Aliquots were removed at the indicated times and spread onto YPD plates in triplicate. Numbers of viable colonies were determined after 2–3 d. (C) The accumulation of prAPI in cvt9Δ can be partially reversed by nitrogen starvation. The apg7Δ (VDY101), cvt3 (THY119), and cvt9Δ (AHY001) strains were grown to midlog phase and shifted to SD-N. Protein extracts were prepared at the indicated times and analyzed by immunoblots with antiserum to API. (D) ALP activity assay for autophagy. Wild-type (TN125), apg1Δ (YYK126), and cvt9Δ (YYK127) cells expressing Pho8Δ60 were shifted from nutrient-rich medium (white bars) to SD-N nitrogen-depleted medium (black bars) for 4 h. The level of autophagy induction was determined by an ALP activity assay as described in Materials and Methods. The graph is the average of three experiments. The cvt9Δ strain is not defective in the vacuolar delivery of the autophagy marker Pho8Δ60 during starvation conditions. (E) Cvt9 is not required for the formation of autophagosomes. The cvt9Δ (YYK127) strain was grown in nutrient-rich (YPD) and nitrogen starvation (SD-N) conditions supplemented with the protease inhibitor PMSF and examined by electron microscopy as described in Materials and Methods. In YPD, Cvt complexes (indicated by an arrow) could be detected in the cytoplasm. In SD-N, cvt9Δ cells accumulated autophagic bodies in the vacuole when treated with PMSF.
Mentions: The CVT9 single-copy plasmid rescued the prAPI processing defect in the cvt9 mutant strain (Fig. 1 A), although overexpression of Cvt9 using a multicopy CVT9 plasmid resulted in a moderate accumulation of prAPI. Although the CVT9 clone was isolated using the nitrogen starvation strategy, the viability of the cvt9- strain remained relatively robust in starvation conditions when compared with a typical mutant in the autophagy pathway (e.g., cvt10/apg1; Scott et al. 1996; Fig. 1 B). Viability under starvation conditions reflects a capacity for autophagy, indicating an ability to recycle cytosolic material after delivery to the vacuole. Therefore, we next determined if the resistance to nitrogen starvation exhibited by the cvt9Δ strain reflects its ability to carry out autophagy.

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