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First published online 20 February 2007
doi: 10.1242/jcs.002717

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Differential detergent resistance of the apical and basolateral NPPases: relationship with polarized targeting

¹ INSERM UMR S 538, Paris, France
² Université Pierre et Marie Curie, Paris, France
³ Monash University, Prahran, Victoria, Australia

View larger version (17K): [in this window] [in a new window]   Fig. 1. Schematic representation of wild-type proteins, mutants and constructs. The transmembrane and extracellular domains of NPP3 and NPP1 are drawn in black and white, respectively. The amino acid sequences of the cytoplasmic tails are given in single letter code. Amino acids altered by site-directed mutagenesis are underlined. In the NPP3-AAASLLAP construct, the sequence AAASLLAP of NPP1 was inserted after the second amino acid at the N terminus of NPP3. Constructs are not drawn to scale.

View larger version (75K): [in this window] [in a new window]   Fig. 2. NPP3 and NPP1 differ with respect to their solubility in cold non-ionic detergents. (A,B) Scanning confocal micrographs of MDCK cells stably transfected with NPP3 (A), or NPP1 (B) cDNAs. NPP3 and NPP1 were labeled with specific monoclonal antibodies followed by secondary FITC-conjugated antibodies. Projections (xy and xz views) are shown. Bars, 10 µm. (C,D) DRM association of NPP3 and NPP1. MDCK-NPP3 and MDCK-NPP1 cell homogenates were extracted with 0.5% Triton X-100, or 0.5% Lubrol WX, or 0.5% Brij 58 at 4°C, and separated on sucrose gradients. The distribution of NPP3 (C) and NPP1 (D) was analyzed by immunoblotting after immunoprecipitation from each fraction. Typical immunoblots are shown. Bar charts show quantification of the DRM-associated protein (fractions 3-6) (DRM) and soluble protein (fractions 7-12) (Sol) from the blots. Results are means ± s.d. of at least three independent experiments.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effect of cholesterol depletion on DRM association of NPP3. MDCK cells stably transfected with NPP3-WT cDNA were labeled with [3H]cholesterol for 20 hours, then treated (MCD) or mock-treated (mock) with 10 mM methyl--cyclodextrin for 30 minutes before detergent extraction. (A,B) Distribution of NPP3 along the sucrose gradient fractions after solubilization with Lubrol WX (A) or Triton X-100 (B). Cell homogenates were detergent-extracted and separated on sucrose gradients and the distribution of NPP3 was analyzed as in Fig. 2. The bar charts show quantification of the DRM-associated (fractions 3-6; DRM) and soluble proteins (fractions 7-12; Sol). (C) Cholesterol content of Lubrol WX and Triton X-100 DRMs. The amount of cholesterol in the gradient fractions was quantified by counting the radioactivity of pooled aliquots of the DRM and soluble fractions by liquid scintillation.

View larger version (57K): [in this window] [in a new window]   Fig. 4. Apical localization does not correlate with DRM association. (A-C) Scanning confocal micrographs of MDCK cells stably transfected with cDNAs of indicated constructs. Cells were treated as in Fig. 2. Bars, 10 µm. (D-F) DRM association of NPP3-AAASLLAP (D), NPP1-34 (E) and NPP1-AA (F). Cell homogenates were extracted and separated on sucrose gradients and the distribution of the proteins was analyzed as in Fig. 2. Typical immunoblots are shown. Bar chart values are means ± s.d. of at least three independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Resistance of NPP3 to Lubrol extraction is acquired early, independently of targeting. MDCK cells stably expressing NPP3-WT or NPP3-AAASLLAP were labeled for 1 hour with [35S]Met-[35S]Cys. Cells were lysed with Lubrol WX and separated on sucrose gradients. Each fraction was subjected to immunoprecipitation with anti-NPP3 antibody, then immunoprecipitates corresponding to the DRM and soluble fractions (Sol) were separately pooled, and treated (+) or not treated (–) with endo H, separated by SDS-PAGE electrophoresis and analyzed by fluorography. The mature (1), high-mannose (2) and deglycosylated (3) forms are indicated by arrows. The mature forms of both NPP3-WT and NPP3-AAASLLAP (band 1) migrate slightly faster after endo H treatment because not all glycan chains are complex glycosylated (Maurice et al., 1994).

View larger version (64K): [in this window] [in a new window]   Fig. 6. The cytoplasmic domain of NPP3 is responsible for Lubrol-DRM association. (A-C) Scanning confocal micrographs of MDCK cells stably transfected with cDNAs of the indicated constructs. Cells were treated as in Fig. 2. Bars, 10 µm. (D-F) DRM association of NPP1-TmNPP3 (D), NPP3-CytoNPP1 (E), and NPP1-CytoNPP3 (F). Cell homogenates were extracted and floated on sucrose gradients and the distribution of the proteins was analyzed as in Fig. 2. Typical immunoblots are shown. Bar chart values are means ± s.d. of at least three determinations.

View larger version (38K): [in this window] [in a new window]   Fig. 7. Mutation of lysines 14-15 reduces affinity of NPP3 to Lubrol DRMs. (A) scanning confocal micrographs picture of MDCK cells stably expressing NPP3-KK/AA. Cells were labeled with specific monoclonal anti-NPP3 antibody followed by secondary FITC-conjugated antibody. Bar, 10 µm. (B) DRM association of NPP3-KK/AA. Cell homogenates were extracted and separated on sucrose gradients and the distribution of the proteins was analyzed as in Fig. 2. Typical immunoblots are shown. Bar chart values are means ± s.d. of two determinations.