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Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin.

Farr GA, Hull M, Stoops EH, Bateson R, Caplan MJ - Mol. Biol. Cell (2015)

Bottom Line: These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase.Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19 °C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network.Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8026.

No MeSH data available.


Related in: MedlinePlus

Endogenous E-cadherin is not retained in the Golgi complex during an incubation at 19°C. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for the indicated times at various temperatures. Samples were subjected to cell surface biotinylation and then lysed in Triton X-100 buffer. (A) Extracts were precipitated with anti–E-cadherin antibodies, washed, and eluted with 1% SDS before analysis by 8% SDS–PAGE (Lysates). Next 1% SDS eluates were diluted 10-fold, precipitated with streptavidin-agarose, and analyzed as before (Cell Surface). (B) Identical extracts from A were precipitated with antibodies directed against the sodium pump and processed as described. (C) Autoradigraphs from A and B were scanned by densitometry and normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Representative data from two independent experiments. (D) Data analysis was performed as in C and normalized relative to the maximal expression obtained at 37°C.
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Figure 5: Endogenous E-cadherin is not retained in the Golgi complex during an incubation at 19°C. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for the indicated times at various temperatures. Samples were subjected to cell surface biotinylation and then lysed in Triton X-100 buffer. (A) Extracts were precipitated with anti–E-cadherin antibodies, washed, and eluted with 1% SDS before analysis by 8% SDS–PAGE (Lysates). Next 1% SDS eluates were diluted 10-fold, precipitated with streptavidin-agarose, and analyzed as before (Cell Surface). (B) Identical extracts from A were precipitated with antibodies directed against the sodium pump and processed as described. (C) Autoradigraphs from A and B were scanned by densitometry and normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Representative data from two independent experiments. (D) Data analysis was performed as in C and normalized relative to the maximal expression obtained at 37°C.

Mentions: As expected from the results depicted in Figure 3, extracts from the initial pulse period contained primarily the 135-kDa precursor form of E-cadherin (Figure 5A). This protein was converted to the 120-kDa mature form of E-cadherin during the chase at 37°C, as previously reported (Shore and Nelson, 1991). Very little cleavage was detected when samples were incubated at 14°C for up to 4 h, whereas relatively slow cleavage occurs at 19°C, with full processing being completed after 3 h of incubation at this temperature. Of interest, blockade of E-cadherin processing also correlates with decreased E-cadherin degradation. At both 37 and 19°C, the amount of mature E-cadherin decreases during the intervals 30–90 min and 2–4 h, respectively, whereas this disappearance is not observed at 14°C. Although sodium pump synthesis and stability do not appear to be affected under any of the conditions tested, this experiment reveals that the processing of the sugars attached to the Na,K-ATPase β subunit to produce the fully mature 55-kDa form of the protein is inhibited at 14°C (Figure 5B).


Dual pulse-chase microscopy reveals early divergence in the biosynthetic trafficking of the Na,K-ATPase and E-cadherin.

Farr GA, Hull M, Stoops EH, Bateson R, Caplan MJ - Mol. Biol. Cell (2015)

Endogenous E-cadherin is not retained in the Golgi complex during an incubation at 19°C. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for the indicated times at various temperatures. Samples were subjected to cell surface biotinylation and then lysed in Triton X-100 buffer. (A) Extracts were precipitated with anti–E-cadherin antibodies, washed, and eluted with 1% SDS before analysis by 8% SDS–PAGE (Lysates). Next 1% SDS eluates were diluted 10-fold, precipitated with streptavidin-agarose, and analyzed as before (Cell Surface). (B) Identical extracts from A were precipitated with antibodies directed against the sodium pump and processed as described. (C) Autoradigraphs from A and B were scanned by densitometry and normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Representative data from two independent experiments. (D) Data analysis was performed as in C and normalized relative to the maximal expression obtained at 37°C.
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Related In: Results  -  Collection

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Figure 5: Endogenous E-cadherin is not retained in the Golgi complex during an incubation at 19°C. Cells were pulse labeled with [35S]methionine/cysteine for 15 min and chased for the indicated times at various temperatures. Samples were subjected to cell surface biotinylation and then lysed in Triton X-100 buffer. (A) Extracts were precipitated with anti–E-cadherin antibodies, washed, and eluted with 1% SDS before analysis by 8% SDS–PAGE (Lysates). Next 1% SDS eluates were diluted 10-fold, precipitated with streptavidin-agarose, and analyzed as before (Cell Surface). (B) Identical extracts from A were precipitated with antibodies directed against the sodium pump and processed as described. (C) Autoradigraphs from A and B were scanned by densitometry and normalized relative to the highest value for E-cadherin and the Na,K-ATPase. Representative data from two independent experiments. (D) Data analysis was performed as in C and normalized relative to the maximal expression obtained at 37°C.
Mentions: As expected from the results depicted in Figure 3, extracts from the initial pulse period contained primarily the 135-kDa precursor form of E-cadherin (Figure 5A). This protein was converted to the 120-kDa mature form of E-cadherin during the chase at 37°C, as previously reported (Shore and Nelson, 1991). Very little cleavage was detected when samples were incubated at 14°C for up to 4 h, whereas relatively slow cleavage occurs at 19°C, with full processing being completed after 3 h of incubation at this temperature. Of interest, blockade of E-cadherin processing also correlates with decreased E-cadherin degradation. At both 37 and 19°C, the amount of mature E-cadherin decreases during the intervals 30–90 min and 2–4 h, respectively, whereas this disappearance is not observed at 14°C. Although sodium pump synthesis and stability do not appear to be affected under any of the conditions tested, this experiment reveals that the processing of the sugars attached to the Na,K-ATPase β subunit to produce the fully mature 55-kDa form of the protein is inhibited at 14°C (Figure 5B).

Bottom Line: These experiments reveal that E-cadherin is delivered to the cell surface substantially faster than is the Na,K-ATPase.Furthermore, the surface delivery of newly synthesized E-cadherin to the plasma membrane was not prevented by the 19 °C temperature block that inhibits the trafficking of most proteins, including the Na,K-ATPase, out of the trans-Golgi network.Consistent with these distinct behaviors, populations of newly synthesized E-cadherin and Na,K-ATPase become separated from one another within the trans-Golgi network, suggesting that they are sorted into different carrier vesicles that mediate their post-Golgi trafficking.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520-8026.

No MeSH data available.


Related in: MedlinePlus