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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.

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Inactivation of RE caused an inhibition of VSV-G transport in the secretory pathway. MDCKT cells were prepared identically as in Fig. 5 but processed for immunofluorescence every 30 min up to 2.5 h total time of chase at 31°C. Peroxide was not present in control cells (A), whereas samples whose REs were inactivated were subject to peroxide (B). Cells in B that apparently escaped the DAB inactivation at long times of chase are marked by asterisks. Red, surface VSV-G (TKG staining); green, total VSV-G fluorescence.
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fig6: Inactivation of RE caused an inhibition of VSV-G transport in the secretory pathway. MDCKT cells were prepared identically as in Fig. 5 but processed for immunofluorescence every 30 min up to 2.5 h total time of chase at 31°C. Peroxide was not present in control cells (A), whereas samples whose REs were inactivated were subject to peroxide (B). Cells in B that apparently escaped the DAB inactivation at long times of chase are marked by asterisks. Red, surface VSV-G (TKG staining); green, total VSV-G fluorescence.

Mentions: To ensure that the VSV-G trapped intracellularly was blocked on the way out, we performed a time course in which cells were assayed for surface and total VSV-G at various times after the inactivation step. As shown in Fig. 6, in control cells (Fig. 6 A) the expected ER-like pattern of intracellular VSV-G became a more punctate Golgi-endosomal pattern within 30 min after shifting from 40 to 31°C, although surface VSV-G was not detected until 60–90 min. In cells subjected to DAB inactivation before the temperature shift (Fig. 6 B), the intracellular punctate pattern again appeared within 30 min, and remained in most cells for the remainder of the chase. As expected, little VSV-G appeared at the plasma membrane, with this occurring largely in cells that failed to accumulate VSV-G intracellularly (Fig. 6 B, asterisk). Such cells either did not take up Tfn-HRP initially or, more likely, reversed the DAB inactivation because surface VSV-G was only observed at long chase times (>120 min).


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)

Inactivation of RE caused an inhibition of VSV-G transport in the secretory pathway. MDCKT cells were prepared identically as in Fig. 5 but processed for immunofluorescence every 30 min up to 2.5 h total time of chase at 31°C. Peroxide was not present in control cells (A), whereas samples whose REs were inactivated were subject to peroxide (B). Cells in B that apparently escaped the DAB inactivation at long times of chase are marked by asterisks. Red, surface VSV-G (TKG staining); green, total VSV-G fluorescence.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Inactivation of RE caused an inhibition of VSV-G transport in the secretory pathway. MDCKT cells were prepared identically as in Fig. 5 but processed for immunofluorescence every 30 min up to 2.5 h total time of chase at 31°C. Peroxide was not present in control cells (A), whereas samples whose REs were inactivated were subject to peroxide (B). Cells in B that apparently escaped the DAB inactivation at long times of chase are marked by asterisks. Red, surface VSV-G (TKG staining); green, total VSV-G fluorescence.
Mentions: To ensure that the VSV-G trapped intracellularly was blocked on the way out, we performed a time course in which cells were assayed for surface and total VSV-G at various times after the inactivation step. As shown in Fig. 6, in control cells (Fig. 6 A) the expected ER-like pattern of intracellular VSV-G became a more punctate Golgi-endosomal pattern within 30 min after shifting from 40 to 31°C, although surface VSV-G was not detected until 60–90 min. In cells subjected to DAB inactivation before the temperature shift (Fig. 6 B), the intracellular punctate pattern again appeared within 30 min, and remained in most cells for the remainder of the chase. As expected, little VSV-G appeared at the plasma membrane, with this occurring largely in cells that failed to accumulate VSV-G intracellularly (Fig. 6 B, asterisk). Such cells either did not take up Tfn-HRP initially or, more likely, reversed the DAB inactivation because surface VSV-G was only observed at long chase times (>120 min).

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