Tyrosine motifs are required for prestin basolateral membrane targeting

Abstract

Prestin is targeted to the lateral wall of outer hair cells (OHCs) where its electromotility is critical for cochlear amplification. Using MDCK cells as a model system for polarized epithelial sorting, we demonstrate that prestin uses tyrosine residues, in a YXXΦ motif, to target the basolateral surface. Both Y520 and Y667 are important for basolateral targeting of prestin. Mutation of these residues to glutamine or alanine resulted in retention within the Golgi and delayed egress from the Golgi in Y667Q. Basolateral targeting is restored upon mutation to phenylalanine suggesting the importance of a phenol ring in the tyrosine side chain. We also demonstrate that prestin targeting to the basolateral surface is dependent on AP1B (μ1B), and that prestin uses transferrin containing early endosomes in its passage from the Golgi to the basolateral plasma membrane. The presence of AP1B (μ1B) in OHCs, and parallels between prestin targeting to the basolateral surface of OHCs and polarized epithelial cells suggest that outer hair cells resemble polarized epithelia rather than neurons in this important phenotypic measure.

Keywords: Cell polarity; Golgi; Hair cell; Protein sorting; Tyrosine.

Fig. 1.. Prestin in mouse outer hair cells is localized along the lateral wall of the cell along with β-catenin and Na/K ATPase.

Shown are cartoons of the organ of Corti (A) and its contained outer hair cells. TM, tectorial membrane; BM Basilar membrane; IHC, inner hair cell; OHC Outer hair cell. (B) A model of an outer hair cell in which prestin is shown lining its lateral wall (green). (C–G) The figure shows serial X-Y sections of mouse outer hair cells labeled with an anti-prestin antibody and then visualized using a Leica gated STED microscope. The sections start at the apical end (left) and end at the basal end (right). There is an uniform labeling of prestin along the lateral wall of the cell (middle three panels). Prestin labeling at the apical end of the cell tapers (C). Similarly, there is a patchy clustering of prestin at the basal pole of the cell (G). Mouse outer hair cells immunostained with antibodies to the basolateral markers β-catenin (H) and Na/K ATPase (I), and demonstrates the localization of these proteins (blue) along the lateral wall of the cell. The cells were counterstained with phalloidin Alexa 546 (red), which shows the presence of the sub cortical lattice of actin along the lateral wall of the cell. (J) The AP1µ1B subunit (green) is present in outer hair cells evidenced by antibody labeling of these cells. The figure shows co labeling of these cells with Na/K ATPase (red). Scale bar is 10 microns. These experiments were repeated five times.

Fig. 2.. Y520 and Y667 contained within the YXXΦ motif are important for basolateral targeting of prestin in MDCK cells.

The figure shows MDCK cells transiently transfected with wild type prestin and mutations of prestin: Y520Q, Y526Q, Y616Q and Y667Q. The cells were fixed after 36 hours. Prestin was tagged with YFP at its C-terminus (green) and the cells were stained with an antibody to β-catenin. Shown are serial X-Y confocal sections along the z axis (A). The corresponding X-Z sections are shown at the bottom. Mutations at Y520 and Y667 result in a failure to target the basolateral surface of the cell along with intracellular retention of the protein and apical trafficking. (B,C) Pearson's correlation of prestin with β-catenin and a ratio of prestin fluorescence on the surface of the cell compared to the total in the cell. The mean Pearson's correlation values were: wt 0.23 (+/−0.028SE, n = 7); Y520Q −0.045 (+/−0.016 SE, n = 7); Y526Q 0.17 (+/−0.025 SE, n = 14); Y616Q 0.175 (+/−0.033 SE, n = 14); Y667Q 0.01386A (+/−.03 SE, n = 10). The differences between wt and Y520Q and Y667Q were considered significant. A one way ANOVA (parametric) with Bonferroni post test comparison yielded a p value of <0.001 for wt vs Y520Q and wt vs Y667Q; wt vs Y616Q and Y667Q were not significant. The mean surface to total ratios were: wt 0.67 (+/−0.016 SE, n = 22); Y520Q −0.39 (+/−0.0129 SE, n = 20); Y526Q 0.78 (+/−0.013 SE, n = 13); Y616Q 0.73 (+/−0.02 SE, n = 13); Y667Q 0.40 (+/−.044 SE, n = 13). Here too the differences between wt and Y520Q and Y667Q were significant. A one-way ANOVA (parametric) with Bonferroni post-test comparison yielded a p value of <0.001 for wt vs Y520Q and wt vs Y667Q. The scale bar is 10 microns.

Fig. 3.. Mutation of Y520 and Y667 result in increased delivery of prestin to the apical surface of MDCK cells.

MDCK cells transiently transfected with wt prestin YFP and the two constructs Y520Q prestin YFP, and Y667Q prestin YFP were fixed at 36 hours and stained with antibody to the apical marker GP130 (podohexin). There is significant apical targeting of Y520Q and Y667Q evident in the serial X-Y sections along the z axis and the corresponding X-Z sections at the bottom. The bottom panel shows Pearson's correlation of prestin YFP and GP130 confirming absent apical targeting of the wild type construct and apical targeting of Y520Q and Y667Q. The mean Pearson's correlation values were: wt −0.027 (+/−0.011 SE, n = 7); Y520Q 0.105 (+/−0.035 SE, n = 5); Y667Q 0.17 (+/−0.063 SE, n = 11). The differences between wt prestin and Y520Q and wt prestin and Y667Q were significant. A one-way ANOVA (parametric) yielded a p value of <0.01 between wt prestin and Y520Q, and a p value of <0.001 between wt prestin and Y667Q. The scale bar is 5 microns.

Fig. 4.. Mutation of Y520Q and Y667Q results in targeting of prestin to the plasma membrane of HEK cells, and presence of NLC in Y667Q, Y520F and Y667F.

The upper panels show HEK cells transfected with wild type prestin-YFP, and the two constructs Y520Q prestin YFP, and Y667Q prestin YFP that were fixed 48 hours after transfection. Cells were stained with antibodies to Na/K ATPase and visualized by confocal microscopy. Wild type prestin YFP, Y520Q prestin YFP and Y667Q prestin YFP all target the plasma membrane as evidenced by its co-localization with plasma membrane Na/K ATPase. The lower panel shows NLC traces of different mutations at Y520 and Y667. The actual values of NLC parameters along with cell numbers are given in supplementary material Table S1. The scale bar is 10 microns.

Fig. 5.. The phenol ring in the tyrosine residue is critical for the targeting of prestin to the basolateral surface of MDCK cells.

MDCK cells transiently transfected with prestin YFP and the mutations Y520Q, Y520A, Y520S and Y520F were fixed 36 hours after transfection and imaged by confocal microscopy. The basolateral wall of the cell was visualized by immunostaining with anti-β-catenin antibody. The mutations Y520A, Y520Q, and Y520S failed to target the basolateral surface of the cell, while Y520F was targeted to the basolateral surface of the cell. Pearson's correlation between prestin YFP and β–catenin confirm the observed co-localization of wild type prestin-YFP with β-catenin and Y520F with β-catenin. The mean Pearson's correlation values were: wt prestin 0.34 (+/−0.033 SE, n = 8); Y520A 0.019 (+/−0.10 SE, n = 5); Y520Q 0.002 (+/−0.052 SE, n = 5); Y520S −0.05 (+/−0.03, n = 6); Y520F 0.25 (+/−0.045 SE, n = 6). A one way ANOVA revealed significant differences between wt prestin and Y520A (P<0.01), wt prestin and Y520Q (P<0.001), and wt prestin and Y520S (P<0.001). The differences between wt prestin and Y520F were not significant. The scale bar is 10 microns.

Fig. 6.. Egress from the Golgi is poor/absent in Y520Q and delayed in Y667Q.

MDCK cells transiently transfected with prestin YFP, and the mutations Y520Q and Y667Q contained in the prestin YFP cassette were fixed after 12, 20, 30 and 40 hours after transfection. The cells were then stained with antibodies to the Golgi protein Giantin. While prestin YFP exits the Golgi at 20 hours, Y520 dos not egress from the Golgi and Y667Q leaves the Golgi after 40 hours. A graphical form of Pearson's correlation between prestin YFP and Giantin at the different time points is shown below. For wt prestin there was a significant difference on one way ANOVA between the Pearson's correlation between prestin and Giantin at all times compared to 12 hours: 12 hours vs 20 hours, P<0.05; 12 hours vs 30 hours, P<0.001; and 12 hours vs 40 hours P<0.001. In contrast, the differences in Pearson's correlation between Y520Q and Giantin at all times compared to 12 hours were not significantly different (P>0.05 on one way ANOVA). For Y667Q the Pearson's correlation with Giantin was significantly different on one way ANOVA between 12 hours and 40 hours (P<0.05), while the remaining two time points were not significantly different (12 hours vs 20 hours, P>0.05; 12 hours vs 30 hours, P>0.05). The mean values for the different time points were as follows: wt prestin 12, 20, 30 and 40 hours: 0.23 (+/−0.08 SE, n = 5), 0.09 (+/−0.04 SE, n = 10), −0.06 (+/−0.03 SE, n = 12), −0.162 (+/−0.02 SE, n = 13); Y520Q 12, 20, 30 and 40 hours: 0.17 (+/−0.02 SE, n = 5), 0.114 (+/−0.04 SE, n = 6), 0.145 (+/−0.06 SE, n = 13), 0.08 (+/−0.02 SE, n = 8); Y667Q 12, 20, 30 and 40 hours: 0.112 (0.03 SE, n = 13), 0.04 (+/−0.02 SE, n = 9); 0.026 (+/−0.05 SE, n = 8) and −0.04(+/−0.03 SE, n = 8). The scale bar is 10 microns.

Fig. 7.. Prestin YFP uses transferrin containing endosomes to transit from the Golgi to the basolateral membrane.

MDCK cells were transiently transfected with prestin-YFP and kept at 19°C to induce Golgi block. Cells were incubated with transferrin - Alexa 647 for 10 minutes. The incubation temperature was raised to 37°C after which cells were fixed at 0, 1 minute, 5 minutes, 10 minutes and 15 minutes. Cells were then imaged with confocal microscopy. Shown are confocal images in the X-Y plane at the basolateral Z axis of the cell that were fixed at different times after raising the temperature to 37°C. With increasing time there is co-localization of prestin-YFP exiting the Golgi with transferring - Alexa 647. The right hand panels shows merged and individual Alexa 647 and prestin-YFP images of an enlarged area at 15 minutes after raising the temperature to 37°C. Co-localization of prestin YFP and transferrin 647 is demonstrated. The scale bar is 10 microns.

Fig. 8.. Targeting of prestin YFP to the basolateral membrane requires AP1B (μ1B).

MDCK cells were electroporated with prestin YFP plasmid and siRNA to AP1B (μ1B). Cells were plated at confluent density and fixed after 30 hours. Cells were stained with antibodies to the apical marker GP130 and visualized by confocal microscopy. Shown are X-Y images along the z axis of transfected cells. The lowest panel shows the corresponding X-Z sections. Transfection of MDCK cells with siRNA to AP1B (μ1B) resulted in an apical localization of prestin YFP and near absence in targeting to the basolateral surface. In contrast, cells co-transfected with control siRNA (chicken KCNMB4) resulted in basolateral targeting of prestin YFP. The scale bar is 10 microns.