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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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Membrane-associated API is imported into the vacuole  as an oligomer. The K12R API mutant was pulse labeled for 10  min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20–40-min  window of the 30°C shift period and lysed with glass beads, and  the resulting extract was separated by 20–50% glycerol gradients.  Fractions were collected, immunoprecipitated with antiserum to  API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating  with the thyroglobulin molecular mass standard (669 kD).
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Figure 6: Membrane-associated API is imported into the vacuole as an oligomer. The K12R API mutant was pulse labeled for 10 min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20–40-min window of the 30°C shift period and lysed with glass beads, and the resulting extract was separated by 20–50% glycerol gradients. Fractions were collected, immunoprecipitated with antiserum to API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating with the thyroglobulin molecular mass standard (669 kD).

Mentions: A key question that we wanted to address was the oligomeric nature of precursor API during the transport step. The K12R API bound to the membrane at the nonpermissive temperature represented a synchronized population of labeled precursor, making it possible to follow its import into the vacuole upon reversal of the accumulation phenotype. Cells were labeled for 10 min and chased for 30 min at 38°C to accumulate precursor API oligomers on the membrane (Fig. 5 A). The labeled cells were then shifted to 30°C to reverse the block and allow precursor API to chase into the vacuole. The oligomeric state of API was examined during the 20–40 min window of the shift phase, when nearly half of the accumulated precursor API was chased into the vacuole and processed. Samples were removed at 20, 27, 34, and 40 min of the 30°C shift period and analyzed by glycerol gradients and immunoprecipitation. As expected, at the 20-min shift point all of the API is present as the precursor form, and by 40 min we see approximately half of the protein as the mature species (Fig. 6). At all time points, however, we detected only the oligomeric form of API, either as the precursor or mature dodecamer recovered in fraction 7 of the glycerol gradients. The kinetic examination of import indicated that oligomeric precursor API maintained its dodecameric state during the period of transport from the membrane-bound step to entry and processing in the vacuole (Fig. 6).


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)

Membrane-associated API is imported into the vacuole  as an oligomer. The K12R API mutant was pulse labeled for 10  min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20–40-min  window of the 30°C shift period and lysed with glass beads, and  the resulting extract was separated by 20–50% glycerol gradients.  Fractions were collected, immunoprecipitated with antiserum to  API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating  with the thyroglobulin molecular mass standard (669 kD).
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Related In: Results  -  Collection

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Figure 6: Membrane-associated API is imported into the vacuole as an oligomer. The K12R API mutant was pulse labeled for 10 min and chased for 30 min at 38°C and then shifted to 30°C. Samples were removed at the indicated times during the 20–40-min window of the 30°C shift period and lysed with glass beads, and the resulting extract was separated by 20–50% glycerol gradients. Fractions were collected, immunoprecipitated with antiserum to API, and resolved by SDS-PAGE. Both mature (mAPI) and precursor API (prAPI) peaks appeared in fraction 7, cofractionating with the thyroglobulin molecular mass standard (669 kD).
Mentions: A key question that we wanted to address was the oligomeric nature of precursor API during the transport step. The K12R API bound to the membrane at the nonpermissive temperature represented a synchronized population of labeled precursor, making it possible to follow its import into the vacuole upon reversal of the accumulation phenotype. Cells were labeled for 10 min and chased for 30 min at 38°C to accumulate precursor API oligomers on the membrane (Fig. 5 A). The labeled cells were then shifted to 30°C to reverse the block and allow precursor API to chase into the vacuole. The oligomeric state of API was examined during the 20–40 min window of the shift phase, when nearly half of the accumulated precursor API was chased into the vacuole and processed. Samples were removed at 20, 27, 34, and 40 min of the 30°C shift period and analyzed by glycerol gradients and immunoprecipitation. As expected, at the 20-min shift point all of the API is present as the precursor form, and by 40 min we see approximately half of the protein as the mature species (Fig. 6). At all time points, however, we detected only the oligomeric form of API, either as the precursor or mature dodecamer recovered in fraction 7 of the glycerol gradients. The kinetic examination of import indicated that oligomeric precursor API maintained its dodecameric state during the period of transport from the membrane-bound step to entry and processing in the vacuole (Fig. 6).

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