Limits...
Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

Ang AL, Taguchi T, Francis S, Fölsch H, Murrells LJ, Pypaert M, Warren G, Mellman I - J. Cell Biol. (2004)

Bottom Line: Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells.Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface.Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute of Cancer Research, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

Show MeSH

Related in: MedlinePlus

Immunoisolated VSV-G membranes from MDCKT are associated with Tfn after exit from the TGN. (A) Representative Western blot of immunoisolated basolateral VSV-G-YFP containing membranes from VSV-G-YFP–infected MDCKT cells incubated with Alexa 488-Tfn during a 40°C ER block, 20°C TGN block, and 10-min chase at 31°C. Immunoprecipitation of VSV-G was performed using magnetic beads coupled to anti-GFP antibody (beads lanes) and probed for VSV-G (VSV-G row). VSV-G purified membranes were also probed for the presence of Alexa 488-Tfn using anti–Alexa 488 antibody (Tfn lane). This is a representative blot from four experiments. (C) Same as in A but apical mutant VSV-G-G3-YFP was used; representative blot from three experiments. (B and D) Quantification of Tfn associated with VSV-G under the three temperature conditions from A and B.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172492&req=5

fig4: Immunoisolated VSV-G membranes from MDCKT are associated with Tfn after exit from the TGN. (A) Representative Western blot of immunoisolated basolateral VSV-G-YFP containing membranes from VSV-G-YFP–infected MDCKT cells incubated with Alexa 488-Tfn during a 40°C ER block, 20°C TGN block, and 10-min chase at 31°C. Immunoprecipitation of VSV-G was performed using magnetic beads coupled to anti-GFP antibody (beads lanes) and probed for VSV-G (VSV-G row). VSV-G purified membranes were also probed for the presence of Alexa 488-Tfn using anti–Alexa 488 antibody (Tfn lane). This is a representative blot from four experiments. (C) Same as in A but apical mutant VSV-G-G3-YFP was used; representative blot from three experiments. (B and D) Quantification of Tfn associated with VSV-G under the three temperature conditions from A and B.

Mentions: In a representative SDS-PAGE gel from four separate experiments, Fig. 4 A shows that relatively little Tfn was detected with VSV-G+ membranes isolated during the 40°C ER block. In contrast, more Tfn was coimmunoprecipitated with VSV-G membranes isolated following the 20°C chase and, especially, following the 31°C chase. The total recovery of VSV-G isolated at each time point was identical. These results were quantified by image digitization, demonstrating that relative to the amount of Tfn coprecipitated at 40°C, the efficiency of Tfn coprecipitation increased 2.5-fold after the 20°C shift and 5.5-fold after the 31°C shift (Fig. 4 B). These data were consistent with the immuno-EM experiments, which showed a greater than fourfold increase in VSV-G and Tfn colocalization upon release of the 20°C block (Fig. 3).


Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.

Ang AL, Taguchi T, Francis S, Fölsch H, Murrells LJ, Pypaert M, Warren G, Mellman I - J. Cell Biol. (2004)

Immunoisolated VSV-G membranes from MDCKT are associated with Tfn after exit from the TGN. (A) Representative Western blot of immunoisolated basolateral VSV-G-YFP containing membranes from VSV-G-YFP–infected MDCKT cells incubated with Alexa 488-Tfn during a 40°C ER block, 20°C TGN block, and 10-min chase at 31°C. Immunoprecipitation of VSV-G was performed using magnetic beads coupled to anti-GFP antibody (beads lanes) and probed for VSV-G (VSV-G row). VSV-G purified membranes were also probed for the presence of Alexa 488-Tfn using anti–Alexa 488 antibody (Tfn lane). This is a representative blot from four experiments. (C) Same as in A but apical mutant VSV-G-G3-YFP was used; representative blot from three experiments. (B and D) Quantification of Tfn associated with VSV-G under the three temperature conditions from A and B.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172492&req=5

fig4: Immunoisolated VSV-G membranes from MDCKT are associated with Tfn after exit from the TGN. (A) Representative Western blot of immunoisolated basolateral VSV-G-YFP containing membranes from VSV-G-YFP–infected MDCKT cells incubated with Alexa 488-Tfn during a 40°C ER block, 20°C TGN block, and 10-min chase at 31°C. Immunoprecipitation of VSV-G was performed using magnetic beads coupled to anti-GFP antibody (beads lanes) and probed for VSV-G (VSV-G row). VSV-G purified membranes were also probed for the presence of Alexa 488-Tfn using anti–Alexa 488 antibody (Tfn lane). This is a representative blot from four experiments. (C) Same as in A but apical mutant VSV-G-G3-YFP was used; representative blot from three experiments. (B and D) Quantification of Tfn associated with VSV-G under the three temperature conditions from A and B.
Mentions: In a representative SDS-PAGE gel from four separate experiments, Fig. 4 A shows that relatively little Tfn was detected with VSV-G+ membranes isolated during the 40°C ER block. In contrast, more Tfn was coimmunoprecipitated with VSV-G membranes isolated following the 20°C chase and, especially, following the 31°C chase. The total recovery of VSV-G isolated at each time point was identical. These results were quantified by image digitization, demonstrating that relative to the amount of Tfn coprecipitated at 40°C, the efficiency of Tfn coprecipitation increased 2.5-fold after the 20°C shift and 5.5-fold after the 31°C shift (Fig. 4 B). These data were consistent with the immuno-EM experiments, which showed a greater than fourfold increase in VSV-G and Tfn colocalization upon release of the 20°C block (Fig. 3).

Bottom Line: Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells.Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface.Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Ludwig Institute of Cancer Research, Yale University School of Medicine, New Haven, CT 06520, USA.

ABSTRACT
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.

Show MeSH
Related in: MedlinePlus