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Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase.

Cai T, Wang H, Chen Y, Liu L, Gunning WT, Quintas LE, Xie ZJ - J. Cell Biol. (2008)

Bottom Line: Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACT
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

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Analyses of the Golgi pool of Cav1. (A) P-11 and (B) TCN23-19 cells were transfected with Cav1-YFP. After 24 h, cells were fixed and stained for giantin and imaged. (C) P-11 and (D) TCN23-19 cells were immunostained for endogenous Cav1 and giantin. The white square area was enlarged and showed at the top right corner of the merged image to display the colocalization of giantin with Cav1. (E) P-11 and TCN23-19 cells were extracted by cold 0.1% Triton X-100 for 2 min as described in Materials and methods. Both soluble and insoluble fractions were collected and subjected to Western blot detection of α1 and Cav1. A representative blot of three independent experiments is shown. (F) P-11(a and b) and TCN23-19 (c and d) cells were transfected with Cav1-YFP for 3 h, then cells were fixed and stained for giantin (a and c). In b and d, transfected cells were treated with 10 μg/ml cycloheximide (Chx) for 3 h, then stained for giantin. Similar experiments were repeated at least four times. Images were taken by confocal microscope. Bars stand for 5 μm in all images.
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fig7: Analyses of the Golgi pool of Cav1. (A) P-11 and (B) TCN23-19 cells were transfected with Cav1-YFP. After 24 h, cells were fixed and stained for giantin and imaged. (C) P-11 and (D) TCN23-19 cells were immunostained for endogenous Cav1 and giantin. The white square area was enlarged and showed at the top right corner of the merged image to display the colocalization of giantin with Cav1. (E) P-11 and TCN23-19 cells were extracted by cold 0.1% Triton X-100 for 2 min as described in Materials and methods. Both soluble and insoluble fractions were collected and subjected to Western blot detection of α1 and Cav1. A representative blot of three independent experiments is shown. (F) P-11(a and b) and TCN23-19 (c and d) cells were transfected with Cav1-YFP for 3 h, then cells were fixed and stained for giantin (a and c). In b and d, transfected cells were treated with 10 μg/ml cycloheximide (Chx) for 3 h, then stained for giantin. Similar experiments were repeated at least four times. Images were taken by confocal microscope. Bars stand for 5 μm in all images.

Mentions: The decrease in plasma membrane Cav1 in TCN23-19 cells could be due to defects in the Golgi processing of Cav1. To test whether the Golgi exit of Cav1 in the knockdown cells is significantly reduced, we first determined whether perinuclear Cav1-YFP colocalized with giantin, a trans-Golgi marker, and calnexin, an ER marker. As expected, some colocalization between Cav1-YFP and giantin (Fig. 7, A and B), but very few between Cav1-YFP and calnexin (not depicted), was detected. However, a majority of the accumulated perinuclear Cav1-YFP in the knockdown cells apparently resided in vesicles outside of the Golgi complex (Fig. 7 B). This pattern of distribution was also true in YFP-Cav1–expressing cells (not depicted). To rule out the possibility that the different cell lines may make a difference in processing transfected proteins, we then performed the second set of experiments in which we examined the Golgi distribution of endogenous Cav1, showing essentially the same pattern of Cav1 distribution in both types of cells (Fig. 7, C and D). Moreover, it is known that the Golgi pool of caveolin is in low molecular weight oligomers and is soluble in low concentration of Triton X-100 at low temperature (Pol et al., 2005). As depicted in Fig. 7 E, we found that a majority of the Cav1 was insoluble in cold 0.1% Triton X-100 in both control and knockdown cells, and that Na/K-ATPase knockdown did not change the amount of soluble Cav1.


Regulation of caveolin-1 membrane trafficking by the Na/K-ATPase.

Cai T, Wang H, Chen Y, Liu L, Gunning WT, Quintas LE, Xie ZJ - J. Cell Biol. (2008)

Analyses of the Golgi pool of Cav1. (A) P-11 and (B) TCN23-19 cells were transfected with Cav1-YFP. After 24 h, cells were fixed and stained for giantin and imaged. (C) P-11 and (D) TCN23-19 cells were immunostained for endogenous Cav1 and giantin. The white square area was enlarged and showed at the top right corner of the merged image to display the colocalization of giantin with Cav1. (E) P-11 and TCN23-19 cells were extracted by cold 0.1% Triton X-100 for 2 min as described in Materials and methods. Both soluble and insoluble fractions were collected and subjected to Western blot detection of α1 and Cav1. A representative blot of three independent experiments is shown. (F) P-11(a and b) and TCN23-19 (c and d) cells were transfected with Cav1-YFP for 3 h, then cells were fixed and stained for giantin (a and c). In b and d, transfected cells were treated with 10 μg/ml cycloheximide (Chx) for 3 h, then stained for giantin. Similar experiments were repeated at least four times. Images were taken by confocal microscope. Bars stand for 5 μm in all images.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2542476&req=5

fig7: Analyses of the Golgi pool of Cav1. (A) P-11 and (B) TCN23-19 cells were transfected with Cav1-YFP. After 24 h, cells were fixed and stained for giantin and imaged. (C) P-11 and (D) TCN23-19 cells were immunostained for endogenous Cav1 and giantin. The white square area was enlarged and showed at the top right corner of the merged image to display the colocalization of giantin with Cav1. (E) P-11 and TCN23-19 cells were extracted by cold 0.1% Triton X-100 for 2 min as described in Materials and methods. Both soluble and insoluble fractions were collected and subjected to Western blot detection of α1 and Cav1. A representative blot of three independent experiments is shown. (F) P-11(a and b) and TCN23-19 (c and d) cells were transfected with Cav1-YFP for 3 h, then cells were fixed and stained for giantin (a and c). In b and d, transfected cells were treated with 10 μg/ml cycloheximide (Chx) for 3 h, then stained for giantin. Similar experiments were repeated at least four times. Images were taken by confocal microscope. Bars stand for 5 μm in all images.
Mentions: The decrease in plasma membrane Cav1 in TCN23-19 cells could be due to defects in the Golgi processing of Cav1. To test whether the Golgi exit of Cav1 in the knockdown cells is significantly reduced, we first determined whether perinuclear Cav1-YFP colocalized with giantin, a trans-Golgi marker, and calnexin, an ER marker. As expected, some colocalization between Cav1-YFP and giantin (Fig. 7, A and B), but very few between Cav1-YFP and calnexin (not depicted), was detected. However, a majority of the accumulated perinuclear Cav1-YFP in the knockdown cells apparently resided in vesicles outside of the Golgi complex (Fig. 7 B). This pattern of distribution was also true in YFP-Cav1–expressing cells (not depicted). To rule out the possibility that the different cell lines may make a difference in processing transfected proteins, we then performed the second set of experiments in which we examined the Golgi distribution of endogenous Cav1, showing essentially the same pattern of Cav1 distribution in both types of cells (Fig. 7, C and D). Moreover, it is known that the Golgi pool of caveolin is in low molecular weight oligomers and is soluble in low concentration of Triton X-100 at low temperature (Pol et al., 2005). As depicted in Fig. 7 E, we found that a majority of the Cav1 was insoluble in cold 0.1% Triton X-100 in both control and knockdown cells, and that Na/K-ATPase knockdown did not change the amount of soluble Cav1.

Bottom Line: Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface.These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit.Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Toledo College of Medicine, Health Science Campus, Toledo, OH 43614, USA.

ABSTRACT
Here, we show that the Na/K-ATPase interacts with caveolin-1 (Cav1) and regulates Cav1 trafficking. Graded knockdown of Na/K-ATPase decreases the plasma membrane pool of Cav1, which results in a significant reduction in the number of caveolae on the cell surface. These effects are independent of the pumping function of Na/K-ATPase, and instead depend on interaction between Na/K-ATPase and Cav1 mediated by an N-terminal caveolin-binding motif within the ATPase alpha1 subunit. Moreover, knockdown of the Na/K-ATPase increases basal levels of active Src and stimulates endocytosis of Cav1 from the plasma membrane. Microtubule-dependent long-range directional trafficking in Na/K-ATPase-depleted cells results in perinuclear accumulation of Cav1-positive vesicles. Finally, Na/K-ATPase knockdown has no effect on processing or exit of Cav1 from the Golgi. Thus, the Na/K-ATPase regulates Cav1 endocytic trafficking and stabilizes the Cav1 plasma membrane pool.

Show MeSH
Related in: MedlinePlus