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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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Oligomerization kinetics of precursor API. Wild-type  cells were pulse labeled for 2 min at 30°C followed by nonradioactive chase reactions. Aliquots were removed at the indicated  chase times and lysed with glass beads, and the resulting cell extracts were separated on 20–50% glycerol gradients. Fractions  were collected, immunoprecipitated with antiserum to API, and  resolved by SDS-PAGE. Molecular mass standards corresponding to the fractions: 45 kD, fraction 2; 158 kD, fraction 4; 240 kD,  fraction 5; 450 kD, fraction 6; and 669 kD, fraction 7. Quantitation of the radioactive signals was performed using a phosphorimager (model Storm; Molecular Dynamics).
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Figure 2: Oligomerization kinetics of precursor API. Wild-type cells were pulse labeled for 2 min at 30°C followed by nonradioactive chase reactions. Aliquots were removed at the indicated chase times and lysed with glass beads, and the resulting cell extracts were separated on 20–50% glycerol gradients. Fractions were collected, immunoprecipitated with antiserum to API, and resolved by SDS-PAGE. Molecular mass standards corresponding to the fractions: 45 kD, fraction 2; 158 kD, fraction 4; 240 kD, fraction 5; 450 kD, fraction 6; and 669 kD, fraction 7. Quantitation of the radioactive signals was performed using a phosphorimager (model Storm; Molecular Dynamics).

Mentions: After a 2-min pulse without a chase, the majority of labeled precursor API was recovered in fractions 2 and 3, consistent with the size of the precursor API monomer (61 kD; Fig. 2, top panel). Interestingly, even at this early time point, a small peak at fraction 7 corresponding to oligomeric precursor could be detected. After a 3-min chase, the relative distribution of precursor API monomer and oligomer was reversed, with the majority of the labeled precursor API assembling into the oligomeric form and the concomitant depletion of the labeled monomer (Fig. 2, second panel). Oligomerization of labeled precursor API was nearly complete after 6 min of the chase reaction, and no monomer was detected after 10 min of chase (Fig. 2, third and fourth panels, respectively). These data indicate a half-time of oligomerization of ∼2 min. Therefore, the in vivo kinetics of precursor API oligomerization are far more rapid than the half-time of API maturation, suggesting that oligomer assembly is an early step in the import of API into the vacuole.


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)

Oligomerization kinetics of precursor API. Wild-type  cells were pulse labeled for 2 min at 30°C followed by nonradioactive chase reactions. Aliquots were removed at the indicated  chase times and lysed with glass beads, and the resulting cell extracts were separated on 20–50% glycerol gradients. Fractions  were collected, immunoprecipitated with antiserum to API, and  resolved by SDS-PAGE. Molecular mass standards corresponding to the fractions: 45 kD, fraction 2; 158 kD, fraction 4; 240 kD,  fraction 5; 450 kD, fraction 6; and 669 kD, fraction 7. Quantitation of the radioactive signals was performed using a phosphorimager (model Storm; Molecular Dynamics).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139888&req=5

Figure 2: Oligomerization kinetics of precursor API. Wild-type cells were pulse labeled for 2 min at 30°C followed by nonradioactive chase reactions. Aliquots were removed at the indicated chase times and lysed with glass beads, and the resulting cell extracts were separated on 20–50% glycerol gradients. Fractions were collected, immunoprecipitated with antiserum to API, and resolved by SDS-PAGE. Molecular mass standards corresponding to the fractions: 45 kD, fraction 2; 158 kD, fraction 4; 240 kD, fraction 5; 450 kD, fraction 6; and 669 kD, fraction 7. Quantitation of the radioactive signals was performed using a phosphorimager (model Storm; Molecular Dynamics).
Mentions: After a 2-min pulse without a chase, the majority of labeled precursor API was recovered in fractions 2 and 3, consistent with the size of the precursor API monomer (61 kD; Fig. 2, top panel). Interestingly, even at this early time point, a small peak at fraction 7 corresponding to oligomeric precursor could be detected. After a 3-min chase, the relative distribution of precursor API monomer and oligomer was reversed, with the majority of the labeled precursor API assembling into the oligomeric form and the concomitant depletion of the labeled monomer (Fig. 2, second panel). Oligomerization of labeled precursor API was nearly complete after 6 min of the chase reaction, and no monomer was detected after 10 min of chase (Fig. 2, third and fourth panels, respectively). These data indicate a half-time of oligomerization of ∼2 min. Therefore, the in vivo kinetics of precursor API oligomerization are far more rapid than the half-time of API maturation, suggesting that oligomer assembly is an early step in the import of API into the vacuole.

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