Polarized trafficking of multipass membrane proteins in Madin-Darby canine kidney cells likely has limited sorting routes
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Sodium-dependent vitamin C transporters, SVCT1 and SVCT2, are multi-pass membrane proteins sorted to the apical and basolateral membranes in epithelial cells, respectively. Sorting uses N- and C-terminal sequences. Previous studies in our lab showed that substituting the N-terminus from other basolateral-targeted proteins or making C-terminal deletions affects trafficking of SVCT and, results in their accumulation in a stable tubulin-like structure (TLS). The TLS was hypothesized to represent functionally disturbed sorting route(s) for multi-pass membrane proteins. To determine whether these routes are shared by other multi-pass membrane proteins, transport assays using flavone (non-transporter mediated), deoxy-glucose (DOG), and phenylalanine (Phe) as substrates were performed for 4 different cell types: wild type MDCK cells, MDCK cells expressing apical SVCT (AP cells), MDCK cells expressing basolateral SVCT (BL cells), and MDCK cells with EGFP-tagged proteins accumulated in the TLS (TLS cells). No difference was detected among cell types in flavone movement. In contrast AP, BL, and TLS cells exhibited significant declines in DOG transepithelial movement from both AP-to-BL and BL-to-AP directions. Compared with wild type, TLS cells showed less total DOG cellular uptake from basolateral side. When Phe was added to the apical side, no difference was found in transepithelial movement but TLS cells accumulated more total Phe than wild type cells. When Phe was added to the basolateral side, more transepithelial movement was detected in BL cells than in wild type or AP cells. TLS cells had more Na-independent Phe accumulation than AP cells. These results suggested that the sorting of exogenous multi-pass membrane proteins affected the sorting of other unrelated native transporters. Thus, there are likely only limited routes for newly-synthesized multi-pass membrane proteins to be sorted to their destinations.