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Transport of a large oligomeric protein by the cytoplasm to vacuole protein targeting pathway.

Kim J, Scott SV, Oda MN, Klionsky DJ - J. Cell Biol. (1997)

Bottom Line: Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting.A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation.Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, University of California, Davis 95616, USA.

ABSTRACT
Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.

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Models for API import via the Cvt pathway. Precursor  API is synthesized and assembled into dodecamers in the cytoplasm, followed by membrane binding. During the rate-limiting  step of the pathway, both models A and B propose a vesicle-mediated mechanism of API entry into the vacuole followed by the  breakdown of the API-containing vesicles and cleavage of the  API propeptide in a PrB-dependent manner to yield the mature  hydrolase. The role of molecular chaperones and the location of  the initial binding of precursor API remain to be resolved. (A)  Oligomeric precursor API directly binds to the vacuolar or prevacuolar membrane before the vesicle-mediated entry into the  vacuole. (B) Genetic analyses have revealed a large overlap between the autophagy and Cvt pathways. In this model, API is delivered to the vacuole via double-membrane autophagic vesicles.  Upon reaching the vacuole, these vesicles fuse to the vacuolar  membrane, releasing a single-membrane vesicle (autophagic  body) containing precursor API. This is followed by the breakdown of the vesicles and subsequent processing of the hydrolase  to the mature enzyme.
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Figure 7: Models for API import via the Cvt pathway. Precursor API is synthesized and assembled into dodecamers in the cytoplasm, followed by membrane binding. During the rate-limiting step of the pathway, both models A and B propose a vesicle-mediated mechanism of API entry into the vacuole followed by the breakdown of the API-containing vesicles and cleavage of the API propeptide in a PrB-dependent manner to yield the mature hydrolase. The role of molecular chaperones and the location of the initial binding of precursor API remain to be resolved. (A) Oligomeric precursor API directly binds to the vacuolar or prevacuolar membrane before the vesicle-mediated entry into the vacuole. (B) Genetic analyses have revealed a large overlap between the autophagy and Cvt pathways. In this model, API is delivered to the vacuole via double-membrane autophagic vesicles. Upon reaching the vacuole, these vesicles fuse to the vacuolar membrane, releasing a single-membrane vesicle (autophagic body) containing precursor API. This is followed by the breakdown of the vesicles and subsequent processing of the hydrolase to the mature enzyme.

Mentions: In the first model, the precursor oligomer binds directly to the vacuole and enters in an endocytic-like manner (Fig. 7 A). This is followed by the breakdown of the API-containing vesicles and processing of precursor API to the mature hydrolase. Alternatively, oligomeric precursor API may initially bind to a prevacuolar compartment before delivery to the vacuole via transport vesicles. The second model (Fig. 7 B) is based on the significant genetic overlap of the Cvt pathway with autophagy, which suggests that these two routes to the vacuole share largely the same cellular machinery (Harding et al., 1996; Scott et al., 1996). Specifically, the double-membrane autophagic vesicles (autophagosomes) that nonselectively capture and deliver cytoplasmic contents (Baba et al., 1994) to the vacuole may also be used by precursor API for its transport to this organelle. However, unlike autophagy, the delivery of API is constitutive. Thus, a continuous, basal-level supply of autophagic vesicles would be required for the selective delivery of API to the vacuole. In this model (Fig. 7 B), API initially binds to putative cusp-shaped progenitors of the double-membrane autophagosomes (Baba et al., 1994). Upon enclosure and autophagosome formation, API is transported to the vacuole, where the outer vesicle membrane fuses with the vacuole, releasing a single-membrane vesicle into the vacuolar lumen. These vesicles (autophagic bodies) are then degraded in a PrB-dependent manner (Takeshige et al., 1992), exposing the oligomeric precursor API to the vacuolar lumen for processing.


Transport of a large oligomeric protein by the cytoplasm to vacuole protein targeting pathway.

Kim J, Scott SV, Oda MN, Klionsky DJ - J. Cell Biol. (1997)

Models for API import via the Cvt pathway. Precursor  API is synthesized and assembled into dodecamers in the cytoplasm, followed by membrane binding. During the rate-limiting  step of the pathway, both models A and B propose a vesicle-mediated mechanism of API entry into the vacuole followed by the  breakdown of the API-containing vesicles and cleavage of the  API propeptide in a PrB-dependent manner to yield the mature  hydrolase. The role of molecular chaperones and the location of  the initial binding of precursor API remain to be resolved. (A)  Oligomeric precursor API directly binds to the vacuolar or prevacuolar membrane before the vesicle-mediated entry into the  vacuole. (B) Genetic analyses have revealed a large overlap between the autophagy and Cvt pathways. In this model, API is delivered to the vacuole via double-membrane autophagic vesicles.  Upon reaching the vacuole, these vesicles fuse to the vacuolar  membrane, releasing a single-membrane vesicle (autophagic  body) containing precursor API. This is followed by the breakdown of the vesicles and subsequent processing of the hydrolase  to the mature enzyme.
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Related In: Results  -  Collection

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Figure 7: Models for API import via the Cvt pathway. Precursor API is synthesized and assembled into dodecamers in the cytoplasm, followed by membrane binding. During the rate-limiting step of the pathway, both models A and B propose a vesicle-mediated mechanism of API entry into the vacuole followed by the breakdown of the API-containing vesicles and cleavage of the API propeptide in a PrB-dependent manner to yield the mature hydrolase. The role of molecular chaperones and the location of the initial binding of precursor API remain to be resolved. (A) Oligomeric precursor API directly binds to the vacuolar or prevacuolar membrane before the vesicle-mediated entry into the vacuole. (B) Genetic analyses have revealed a large overlap between the autophagy and Cvt pathways. In this model, API is delivered to the vacuole via double-membrane autophagic vesicles. Upon reaching the vacuole, these vesicles fuse to the vacuolar membrane, releasing a single-membrane vesicle (autophagic body) containing precursor API. This is followed by the breakdown of the vesicles and subsequent processing of the hydrolase to the mature enzyme.
Mentions: In the first model, the precursor oligomer binds directly to the vacuole and enters in an endocytic-like manner (Fig. 7 A). This is followed by the breakdown of the API-containing vesicles and processing of precursor API to the mature hydrolase. Alternatively, oligomeric precursor API may initially bind to a prevacuolar compartment before delivery to the vacuole via transport vesicles. The second model (Fig. 7 B) is based on the significant genetic overlap of the Cvt pathway with autophagy, which suggests that these two routes to the vacuole share largely the same cellular machinery (Harding et al., 1996; Scott et al., 1996). Specifically, the double-membrane autophagic vesicles (autophagosomes) that nonselectively capture and deliver cytoplasmic contents (Baba et al., 1994) to the vacuole may also be used by precursor API for its transport to this organelle. However, unlike autophagy, the delivery of API is constitutive. Thus, a continuous, basal-level supply of autophagic vesicles would be required for the selective delivery of API to the vacuole. In this model (Fig. 7 B), API initially binds to putative cusp-shaped progenitors of the double-membrane autophagosomes (Baba et al., 1994). Upon enclosure and autophagosome formation, API is transported to the vacuole, where the outer vesicle membrane fuses with the vacuole, releasing a single-membrane vesicle into the vacuolar lumen. These vesicles (autophagic bodies) are then degraded in a PrB-dependent manner (Takeshige et al., 1992), exposing the oligomeric precursor API to the vacuolar lumen for processing.

Bottom Line: Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting.A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation.Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.

View Article: PubMed Central - PubMed

Affiliation: Section of Microbiology, University of California, Davis 95616, USA.

ABSTRACT
Aminopeptidase I (API) is transported into the yeast vacuole by the cytoplasm to vacuole targeting (Cvt) pathway. Genetic evidence suggests that autophagy, a major degradative pathway in eukaryotes, and the Cvt pathway share largely the same cellular machinery. To understand the mechanism of the Cvt import process, we examined the native state of API. Dodecameric assembly of precursor API in the cytoplasm and membrane binding were rapid events, whereas subsequent vacuolar import appeared to be rate limiting. A unique temperature-sensitive API-targeting mutant allowed us to kinetically monitor its oligomeric state during translocation. Our findings indicate that API is maintained as a dodecamer throughout its import and will be useful to study the posttranslational movement of folded proteins across biological membranes.

Show MeSH
Related in: MedlinePlus