RESUMO
Polycystin-1 (PC1), the product of the polycystic kidney disease-1 (PKD1) gene, has a number of reported missense mutations whose pathogenicity is indeterminate. Previously, we utilized N-linked glycosylation reporter tags along with membrane insertion and topology assays to define the 11 membrane-spanning domains (I-XI) of PC1. In this report, we utilize glycosylation assays to determine whether two reported human polymorphisms/missense mutations within transmembrane (TM) domains VI and X affect the membrane topology of PC1. M3677T within TM VI had no effect on the topology of this TM domain as shown by the ability of two native N-linked glycosylation sites within the extracellular loop following TM VI to be glycosylated. In contrast, G4031D, within TM X, decreased the glycosylation of TM X reporter constructs, demonstrating that the substitution affected the C-terminal translocating activity of TM X. Furthermore, G4031D reduced the membrane association of TM X and XI together. These results suggest that G4031D affects the membrane insertion and topology of the C-terminal portion of polycystin-1 and represents a bona fide pathogenic mutation.
