These results suggest that Tctex-1 is contributed to the vectorial transport of rhodopsin from your TGN to the apical plasma membrane in MDCK cells. We have shown previously the cytoplasmic tail of rhodopsin encodes an autonomous apical sorting transmission in polarized MDCK cells: addition of the COOH-terminal 39 residues of rhodopsin redirected the basolateral membrane protein CD7 to the apical membrane (Chuang and Sung 1998). min at 4C. The high-speed supernatants were then separated by velocity sedimentation on 5C20% linear sucrose gradients (Paschal et al. 1991). 12 fractions of 1 1 mL each were collected from the bottom of the tube, and 20-L samples from each portion were utilized for SDS-PAGE and immunoblotting. The gradients were calibrated with thyroglobulin (19S) and catalase (11.3S) requirements. Domain-selective Surface Biotinylation/Membrane Focusing on Assay For the microtubule depolymerization experiments, a rhodopsin-expressing MDCK stable line cultivated on Transwell filters (Chuang and Sung 1998) was treated with 33 M nocodazole (33 mM stock in DMSO) or 0.1% DMSO at 4C for 30 min followed by incubation at 37C for 3.5 h to depolymerize microtubules. Microtubule depolymerization was confirmed by -tubulin immunostaining of duplicate filters. Nocodazole treatment did not impact MDCK transepithelial resistance (data not demonstrated; Ojakian and Schwimmer 1988; Parczyk et al. 1989). The cells underwent pulseCchase labeling with [35S]cysteine/methionine (New England Nuclear) in the presence of nocodazole or DMSO. At each chase timepoint, cells were chilled on snow and processed for SANT-1 domain-selective surface biotinylation (Chuang and Sung 1998). In brief, glycosylated surface proteins were selectively biotinylated from either the apical or basolateral part using biotin-LC-hydrazide (Pierce Chemical Co.). Cells on excised filters were lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, and protease inhibitors at 4C for 30 min. After centrifugation of the lysate at 14,000 for 15 min, biotinylated rhodopsin was isolated by sequential immunoprecipitation with rhodopsin mAb B6-30 followed by binding to streptavidin-agarose (Pierce Chemical Co.). The biotinylated rhodopsin was then separated by SDS-PAGE, transferred to nitrocellulose, and the amount of radiolabeled rhodopsin was quantitated by phosphorimaging using a Storm scanner (Molecular Dynamics). To measure the surface distribution of rhodopsin in FLAG-RP3Cexpressing cells, T23/FLAG-RP3 cells were plated at low denseness in the presence or absence of doxycycline for 3 d SANT-1 before plating at high denseness (1.5 106 per well) onto 24-mm Transwell filters. After 5 d, polarized monolayers were infected with Ad-CMV-Rho for 24 h followed by biotin-LC-hydrazide surface labeling from either part. Rhodopsin was immunoprecipitated from your cell lysates with B6-30 mAb. Immunoprecipitated rhodopsin was then separated by SDS-PAGE and transferred to a nitrocellulose membrane. Finally, biotinylated rhodopsin was visualized using horseradish peroxidaseCconjugated streptavidin (Kirkegaard and Perry Laboratories) and ECL-Plus (Amersham Pharmacia Biotech) followed by quantitation of chemifluorescence using a Storm scanner. The linearity of this detection system for the amounts of rhodopsin indicated was confirmed using biotinylated protein standards (data not demonstrated). For measurement of rhodopsin focusing on in FLAG-RP3 expressing cells, T23/FLAG-RP3 cells were plated and infected with Ad-CMV-Rho as explained above. The cells were then pulse labeled with [35S]cysteine/methionine for 30 min. At each chase timepoint (0, 1, and 2 h), cells were chilled on snow and processed for domain-selective surface biotinylation as explained above. After centrifugation of the lysate at 14,000 for 15 min, biotinylated rhodopsin was isolated by sequential immunoprecipitation with rhodopsin mAb B6-30 followed by binding to streptavidin-agarose. 10% of the B6-30 immunoprecipitate was retained to correct for filter-to-filter variability in [35S]incorporation into rhodopsin. All samples underwent cleavage of their N-glycans by digestion with N-glycanase (Glyko) to simplify quantitation (Sung et al. 1991). The biotinylated rhodopsin was then separated by SDS-PAGE, transferred to nitrocellulose, and the amount of radiolabeled rhodopsin was quantitated by phosphorimaging. For the study SANT-1 of endogenous apical proteins in MDCK cells, T23/FLAG-RP3 cells were induced/uninduced and polarized on Transwell filters as explained in the preceding paragraph. After 5 d, polarized monolayers were metabolically labeled with [35S]cysteine/methionine from your basolateral part for 4 h, chased for 1 h, and then selectively biotinylated from your apical part using sulfo-NHS-LC-biotin (Pierce Chemical Co.), which reacts with main amines. Doxycycline was included during the labeling and chase periods for uninduced filters. After lysis, biotinylated apical proteins were recovered by streptavidin-agarose precipitation and the total recovery was quantitated by liquid scintillation Rabbit polyclonal to ACD counting. Equal amounts of radiolabeled biotinylated protein.