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. 2007 Aug 1;104(32):12999–13004. doi: 10.1073/pnas.0700163104

Fig. 4.

Fig. 4.

The A322D mutation altered α1 subunit transmembrane topology. (A1 and A2) Schematic diagrams demonstrate that if the α1 subunit's M3 domain inserts into the ER membrane, N365 will be cytoplasmic and cannot be glycosylated (A1), but, if M3 fails membrane insertion, N365 will be within the ER lumen and could be glycosylated (A2). Although M4 was shown to be transmembrane, these experiments do not determine the M4 transmembrane status. In all constructs, the N-terminal glycosylation sites N38 and N138 were mutated to glutamines, and amino acids flanking N365 were mutated (K3654D + N366T) to enhance glycosylation. Mock-transfected cells (m) or cells transfected with wild-type (a) or mutant (d) subunits were [35S]-labeled and fractionated by SDS/PAGE. (B) With N365 intact, mutant, but not wild-type, subunit migrated as two distinct bands (n = 7). With N365 mutated to glutamine, both wild-type and mutant subunits migrated as a single band. (C) Mutant subunits were untreated (−) or treated (+) with, peptide N-glycosidase F before SDS/PAGE. (D) Untagged mutant subunits, but not wild-type subunits, migrated as two bands (n = 4); a protein that coimmunopurified with the untagged proteins and migrated with the same molecular mass as the unglycosylated subunit is marked (→).