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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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Related in: MedlinePlus

FRET analyses of the interaction between Cav1 and Na/K-ATPase α1 subunit. YFP- tagged α1, D371N, or mCBM was transfected together with CFP-Cav1 into TCN23-19 cells. FRET analyses were done as described in Materials and methods. YFP-only and CFP-Cav1–transfected cells were used as a negative control. (A) Images were taken before and after photobleaching. The ROI_1 was bleached at 515 nm with full power. Small regions of plasma membrane in (ROI_2) and out (ROI_3) of the bleached area were selected for measurement, and FRET efficiency was calculated from the measurements using the equation as shown in the table. Bar, 2 μm. (B) Corrected FRET efficiency was calculated from each experiment and values are mean ± SE of 4–5 independent experiments. *, P < 0.05 in comparison to control.
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fig4: FRET analyses of the interaction between Cav1 and Na/K-ATPase α1 subunit. YFP- tagged α1, D371N, or mCBM was transfected together with CFP-Cav1 into TCN23-19 cells. FRET analyses were done as described in Materials and methods. YFP-only and CFP-Cav1–transfected cells were used as a negative control. (A) Images were taken before and after photobleaching. The ROI_1 was bleached at 515 nm with full power. Small regions of plasma membrane in (ROI_2) and out (ROI_3) of the bleached area were selected for measurement, and FRET efficiency was calculated from the measurements using the equation as shown in the table. Bar, 2 μm. (B) Corrected FRET efficiency was calculated from each experiment and values are mean ± SE of 4–5 independent experiments. *, P < 0.05 in comparison to control.

Mentions: We have shown that the Na/K-ATPase can be coimmunoprecipitated with Cav1 and that the purified Na/K-ATPase binds to GST-Cav1 N terminus (Wang et al., 2004). To determine whether the interaction between Na/K-ATPase and Cav1 plays a role in regulating caveolar vesicle trafficking, we first performed fluorescence resonance energy transfer (FRET) analysis to test whether these two proteins have the potential to directly interact in live cells. YFP-α1 was used because our prior experiments showed that it could be expressed and targeted to the plasma membrane as the endogenous α1 (Tian et al., 2006). To test which Cav1 construct could be used for FRET analysis, we first transfected the TCN23-19 cells with either CFP-Cav1/YFP-α1 or Cav1-CFP/YFP-α1, then measured the FRET efficiency. As depicted in Fig. 4 A, both CFP-Cav1 and YFP-α1 were targeted to the plasma membrane. When the plasma membrane signals were analyzed, we found significant FRET between this pair of proteins (Fig. 4, A and B), indicating that the Na/K-ATPase and Cav1 are likely to interact in the plasma membrane. When the same experiment was repeated with Cav1-CFP and YFP-α1, we found that both were also targeted to the plasma membrane (not depicted) and yielded a significant FRET. However, CFP-Cav1/YFP-α1 yielded a higher FRET efficiency than that of Cav1-CFP/YFP-α1 pair (15.5 ± 3.4% vs. 9.2 ± 1.3%, n = 5, P < 0.05). This finding is consistent with the fact that the scaffolding domain and a putative caveolin-binding motif are located at the N termini of both proteins (see next paragraph). Thus, the detected difference in FRET efficiency between the N- and C-terminal tags of Cav1 provides a nice control for validating the FRET analysis. Therefore, the following FRET analyses were conducted with the CFP-Cav1/YFP-α1 pair.


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)

FRET analyses of the interaction between Cav1 and Na/K-ATPase α1 subunit. YFP- tagged α1, D371N, or mCBM was transfected together with CFP-Cav1 into TCN23-19 cells. FRET analyses were done as described in Materials and methods. YFP-only and CFP-Cav1–transfected cells were used as a negative control. (A) Images were taken before and after photobleaching. The ROI_1 was bleached at 515 nm with full power. Small regions of plasma membrane in (ROI_2) and out (ROI_3) of the bleached area were selected for measurement, and FRET efficiency was calculated from the measurements using the equation as shown in the table. Bar, 2 μm. (B) Corrected FRET efficiency was calculated from each experiment and values are mean ± SE of 4–5 independent experiments. *, P < 0.05 in comparison to control.
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fig4: FRET analyses of the interaction between Cav1 and Na/K-ATPase α1 subunit. YFP- tagged α1, D371N, or mCBM was transfected together with CFP-Cav1 into TCN23-19 cells. FRET analyses were done as described in Materials and methods. YFP-only and CFP-Cav1–transfected cells were used as a negative control. (A) Images were taken before and after photobleaching. The ROI_1 was bleached at 515 nm with full power. Small regions of plasma membrane in (ROI_2) and out (ROI_3) of the bleached area were selected for measurement, and FRET efficiency was calculated from the measurements using the equation as shown in the table. Bar, 2 μm. (B) Corrected FRET efficiency was calculated from each experiment and values are mean ± SE of 4–5 independent experiments. *, P < 0.05 in comparison to control.
Mentions: We have shown that the Na/K-ATPase can be coimmunoprecipitated with Cav1 and that the purified Na/K-ATPase binds to GST-Cav1 N terminus (Wang et al., 2004). To determine whether the interaction between Na/K-ATPase and Cav1 plays a role in regulating caveolar vesicle trafficking, we first performed fluorescence resonance energy transfer (FRET) analysis to test whether these two proteins have the potential to directly interact in live cells. YFP-α1 was used because our prior experiments showed that it could be expressed and targeted to the plasma membrane as the endogenous α1 (Tian et al., 2006). To test which Cav1 construct could be used for FRET analysis, we first transfected the TCN23-19 cells with either CFP-Cav1/YFP-α1 or Cav1-CFP/YFP-α1, then measured the FRET efficiency. As depicted in Fig. 4 A, both CFP-Cav1 and YFP-α1 were targeted to the plasma membrane. When the plasma membrane signals were analyzed, we found significant FRET between this pair of proteins (Fig. 4, A and B), indicating that the Na/K-ATPase and Cav1 are likely to interact in the plasma membrane. When the same experiment was repeated with Cav1-CFP and YFP-α1, we found that both were also targeted to the plasma membrane (not depicted) and yielded a significant FRET. However, CFP-Cav1/YFP-α1 yielded a higher FRET efficiency than that of Cav1-CFP/YFP-α1 pair (15.5 ± 3.4% vs. 9.2 ± 1.3%, n = 5, P < 0.05). This finding is consistent with the fact that the scaffolding domain and a putative caveolin-binding motif are located at the N termini of both proteins (see next paragraph). Thus, the detected difference in FRET efficiency between the N- and C-terminal tags of Cav1 provides a nice control for validating the FRET analysis. Therefore, the following FRET analyses were conducted with the CFP-Cav1/YFP-α1 pair.

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