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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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The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the  pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots  were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated  with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated  into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase  K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was  also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right).  The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in  the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction  was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation  phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were  removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window  of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.
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Figure 5: The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right). The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.

Mentions: The oligomerization and membrane-binding steps of API targeting appear early in the overall import process. We next examined the oligomeric nature of API during the remainder of the targeting steps. The membrane binding of precursor API oligomer is followed by import into the vacuole and cleavage of the propeptide by proteinase B (PrB), which yields mature API (Klionsky et al., 1992). To study the oligomeric state of precursor API as it enters the vacuolar lumen from the membrane-bound state, we exploited the unique characteristics of an API-targeting mutant in which the twelfth lysine residue in the predicted amphipathic helix of the propeptide was changed to an arginine. The K12R point mutation rendered API defective for import at nonpermissive temperature, 38°C (Fig. 5 D, middle; Oda et al., 1996), while wild-type kinetics were observed at permissive temperature, 30°C (Fig. 5 D, bottom).


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)

The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the  pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots  were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated  with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated  into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase  K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was  also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right).  The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in  the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction  was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation  phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were  removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window  of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.
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Figure 5: The membrane accumulation phenotype of the K12R API ts mutant is thermally reversible. (A) K12R API accumulates in the pellet fraction at nonpermissive temperature. Spheroplasts were labeled for 10 min at 38°C followed by nonradioactive chase. Aliquots were removed at the indicated chase times and separated into supernatant (sup) and pellet fractions. Samples were immunoprecipitated with antiserum to API, and the radiolabeled signals were quantitated. The percent radiolabeled precursor at a given chase point represents the ratio of precursor from each supernatant or pellet fraction to the total API combined in both fractions. (B) Protease accessibility of K12R API in the supernatant and pellet fractions. Spheroplasts were labeled for 5 min, chased for 30 min at 38°C, and separated into a supernatant and pellet fraction after differential osmotic lysis. The supernatant and pellet fractions were subjected to proteinase K and Triton X-100 as indicated and immunoprecipitated with antiserum to API (left). An aliquot of the recovered pellet fraction was also immunoprecipitated with antisera to the vacuolar marker CPY and the cytosolic marker PGK before protease treatment (right). The percent of marker proteins recovered in the pellet fraction was calculated as described for Fig. 3. (C) Accessibility of K12R API in the pellet fraction to cross-linking with Sulfo-NHS-biotin. Labeled spheroplasts were fractionated exactly as in B. The pellet fraction was cross-linked with Sulfo-NHS-biotin (Biotin-X) in the presence or absence of Triton X-100. API and CPY were recovered by immunoprecipitation followed by precipitation with avidin agarose beads. (D) Thermal reversibility of the K12R membrane-accumulation phenotype. The K12R API mutant was pulse-labeled for 10 min, chased for 30 min at 38°C, and then shifted to 30°C (top). Samples were removed at the indicated times during the shift period at 30°C and lysed with glass beads. The resulting cell extracts were immunoprecipitated with antiserum to API and resolved by SDS-PAGE. The double-headed arrow in the top panel marks the 20–40-min window of time when mature API increases from 10 to 56% during the 30°C shift. The K12R API strain was also pulse labeled, chased, and incubated all at 38°C (middle) or 30°C (bottom) in this experiment.
Mentions: The oligomerization and membrane-binding steps of API targeting appear early in the overall import process. We next examined the oligomeric nature of API during the remainder of the targeting steps. The membrane binding of precursor API oligomer is followed by import into the vacuole and cleavage of the propeptide by proteinase B (PrB), which yields mature API (Klionsky et al., 1992). To study the oligomeric state of precursor API as it enters the vacuolar lumen from the membrane-bound state, we exploited the unique characteristics of an API-targeting mutant in which the twelfth lysine residue in the predicted amphipathic helix of the propeptide was changed to an arginine. The K12R point mutation rendered API defective for import at nonpermissive temperature, 38°C (Fig. 5 D, middle; Oda et al., 1996), while wild-type kinetics were observed at permissive temperature, 30°C (Fig. 5 D, bottom).

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