A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells - PubMed (original) (raw)

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells

Sandra Maday et al. Traffic. 2008 Nov.

Abstract

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.

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Figures

Figure 1

Figure 1. Truncation of the PDZ-binding motif causes apical mislocalization of syndecan-1

A) Polarized MDCK cells stably expressing WT syndecan-1 were fixed, permeabilized, processed for IF for syndecan-1 and gp58, and viewed by confocal microscopy. En face images and X–Z sections are shown. Bars, 10 μm. B) Sequence of the cytoplasmic tail of syndecan-1, with the PDZ-binding motif (blue). The last two residues were deleted in ΔYA. C) Polarized MDCK cells stably expressing ΔYA syndecan-1 were processed as in A. Bars, 10 μm.

Figure 2

Figure 2. Biochemical characterization of ΔYA myc-syndecan-1 mislocalization

A) An myc tag was introduced at the N-terminus, after amino acid 22, five residues after the predicted 17-amino acid signal sequence. B and C) Polarized MDCK cells were infected with adenoviruses encoding either WT or ΔYA myc-syndecan-1 for 20 h. B) Cells were surface labeled for syndecan-1, processed for IF and analyzed by confocal microscopy. Bars, 10 μm. C) Cells were placed on ice and biotin was added to the apical domain (A), the basolateral domain (B) or both the apical and basolateral domains (A+B). Biotin was not added in one sample as a control. Biotinylated domain-specific surface protein was retrieved and processed for immunoblot.

Figure 3

Figure 3. Additional mutations within the PDZ-binding motif cause apical mislocalization

A) Polarized MDCK cells stably expressing the indicated syndecan-1 mutant were surface labeled for syndecan-1, processed for IF and analyzed by confocal microscopy. Bars, 10 μm. B) Polarized MDCK cells stably expressing the indicated syndecan-1 mutant were placed on ice and biotin was added to the apical (A) or basolateral (B) domain. Samples were processed as in Figure 2C and quantified in C. Data represent mean values from two independent experiments and error bars indicate standard deviation.

Figure 4

Figure 4. PDZ-binding motif targets syndecan-1 directly to the basolateral membrane

A) Polarized MDCK cells were infected with adenoviruses encoding WT or ΔYA myc-syndecan-1 for 20 h. Cells were pulsed for 15 min at 37°C with 2 mCi/ml 35S-methionine and 35S-cysteine, and chased for 0–3 h. At each time point, cells were placed on ice and biotinylated on the apical (A) or basolateral (B) domain. Total syndecan-1 protein was immunopre-cipitated and eluted. Biotinylated domain-specific surface syndecan-1 protein was retrieved and processed for SDS–PAGE and autoradiography. B) Quantification of A. Data represent mean values from two independent experiments and error bars indicate standard deviation.

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