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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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API-targeting mutants are not defective in API oligomerization. Strain DYY101 (ape1Δ) harboring a single copy plasmid encoding the propeptide deletions was grown to midlog  phase, lysed with glass beads, and separated on a 20–50% glycerol gradient. The Δ9–11 API deletion mutant shown here is a  representative example of the analysis of the cvt (1 to 17) and  propeptide deletion mutants (Δ3–5, Δ6–8, Δ9–11, Δ12–14, Δ15– 17, Δ18–20, Δ25–27, Δ28–30, Δ31–33, Δ34–36, Δ37–39, Δ40–42,  and Δ2–45). Collected fractions were subjected to Western blotting with antiserum to API and the quantitation of the Western  blots (shown in the graph) was performed as in Fig. 1.
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Figure 4: API-targeting mutants are not defective in API oligomerization. Strain DYY101 (ape1Δ) harboring a single copy plasmid encoding the propeptide deletions was grown to midlog phase, lysed with glass beads, and separated on a 20–50% glycerol gradient. The Δ9–11 API deletion mutant shown here is a representative example of the analysis of the cvt (1 to 17) and propeptide deletion mutants (Δ3–5, Δ6–8, Δ9–11, Δ12–14, Δ15– 17, Δ18–20, Δ25–27, Δ28–30, Δ31–33, Δ34–36, Δ37–39, Δ40–42, and Δ2–45). Collected fractions were subjected to Western blotting with antiserum to API and the quantitation of the Western blots (shown in the graph) was performed as in Fig. 1.

Mentions: The first amphipathic helix of the API propeptide is critical for proper targeting of the enzyme (Oda et al., 1996). Specifically, deletions in this region inhibit API from binding to the membrane fraction, thus preventing subsequent import and processing to the mature form (Oda et al., 1996). We examined whether the targeting defect in these propeptide deletion mutants was caused by an oligomerization defect. Cell extracts were prepared from the DYY101 strain deleted for the gene encoding API (ape1Δ) but harboring a single copy plasmid encoding API with the propeptide deletions. Western blot analysis of glycerol gradient fractions indicated that precursor API in all of the propeptide deletion mutants peaked in fraction 7 (Fig. 4), indicating that it was properly oligomerized. In addition, an API molecule with a deletion of the entire propeptide also formed API oligomers (data not shown). These results indicate that, while the propeptide is necessary for binding to the membrane fraction and subsequent import of API into the vacuole, it does not encode any sequence determinants necessary for API oligomerization. In addition, these results demonstrate that dodecameric precursor API assembly can occur in the absence of membrane binding or transport to the vacuole, in agreement with the kinetic and subcellular fractionation data (Figs. 2 and 3).


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)

API-targeting mutants are not defective in API oligomerization. Strain DYY101 (ape1Δ) harboring a single copy plasmid encoding the propeptide deletions was grown to midlog  phase, lysed with glass beads, and separated on a 20–50% glycerol gradient. The Δ9–11 API deletion mutant shown here is a  representative example of the analysis of the cvt (1 to 17) and  propeptide deletion mutants (Δ3–5, Δ6–8, Δ9–11, Δ12–14, Δ15– 17, Δ18–20, Δ25–27, Δ28–30, Δ31–33, Δ34–36, Δ37–39, Δ40–42,  and Δ2–45). Collected fractions were subjected to Western blotting with antiserum to API and the quantitation of the Western  blots (shown in the graph) was performed as in Fig. 1.
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Related In: Results  -  Collection

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Figure 4: API-targeting mutants are not defective in API oligomerization. Strain DYY101 (ape1Δ) harboring a single copy plasmid encoding the propeptide deletions was grown to midlog phase, lysed with glass beads, and separated on a 20–50% glycerol gradient. The Δ9–11 API deletion mutant shown here is a representative example of the analysis of the cvt (1 to 17) and propeptide deletion mutants (Δ3–5, Δ6–8, Δ9–11, Δ12–14, Δ15– 17, Δ18–20, Δ25–27, Δ28–30, Δ31–33, Δ34–36, Δ37–39, Δ40–42, and Δ2–45). Collected fractions were subjected to Western blotting with antiserum to API and the quantitation of the Western blots (shown in the graph) was performed as in Fig. 1.
Mentions: The first amphipathic helix of the API propeptide is critical for proper targeting of the enzyme (Oda et al., 1996). Specifically, deletions in this region inhibit API from binding to the membrane fraction, thus preventing subsequent import and processing to the mature form (Oda et al., 1996). We examined whether the targeting defect in these propeptide deletion mutants was caused by an oligomerization defect. Cell extracts were prepared from the DYY101 strain deleted for the gene encoding API (ape1Δ) but harboring a single copy plasmid encoding API with the propeptide deletions. Western blot analysis of glycerol gradient fractions indicated that precursor API in all of the propeptide deletion mutants peaked in fraction 7 (Fig. 4), indicating that it was properly oligomerized. In addition, an API molecule with a deletion of the entire propeptide also formed API oligomers (data not shown). These results indicate that, while the propeptide is necessary for binding to the membrane fraction and subsequent import of API into the vacuole, it does not encode any sequence determinants necessary for API oligomerization. In addition, these results demonstrate that dodecameric precursor API assembly can occur in the absence of membrane binding or transport to the vacuole, in agreement with the kinetic and subcellular fractionation data (Figs. 2 and 3).

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