Endoglycosidase H sensitivity and cell surface biotinylation of mutated polycystin-2 proteins. (a) Sequence comparison of polycystin-2 from various species. Shown are amino acids 569–580 of the human protein; the last three amino acids of the presumptive fourth membrane-spanning segment (TMD4) are highlighted in gray. (b) Endoglycosidase H_f (Endo H) assay of HA epitope–tagged full-length polycystin-2 (PC2, HA) and of polycystin-2 (1–703) without additional mutation (No mut.) and with alanine substitutions at positions 572–574 and 575–577. The full-length polycystin-2 protein and the triple-alanine substitution mutants are fully sensitive to treatment with Endo H, but the polycystin-2 (1–703) protein without additional mutations is not (asterisk indicates the resistant band). The band indicated by the arrow probably represents full-length polycystin-2 that did not enter the gel. (c–e) Surface biotinylation of COS-7 (c and e) and LLC-PK₁ cells (d) transiently transfected with expression plasmids for the indicated proteins. The α₁ subunit of the Na⁺/K⁺-ATPase served as a control for efficient biotinylation of plasma membrane proteins. When proteins were (mostly) retained in intracellular membrane compartments, no signal or only a weak signal was detected after precipitation with NeutrAvidin beads. The arrow in d indicates a nonspecific band. Molecular masses (in kD) are indicated on the left of panels b–e. L, whole-cell lysate; B, proteins precipitated with NeutrAvidin beads; S, proteins left in the supernatant after precipitation with NeutrAvidin beads.