ABCMdb

PMID: 10506167

Raab-Graham KF, Cirilo LJ, Boettcher AA, Radeke CM, Vandenberg CA
Membrane topology of the amino-terminal region of the sulfonylurea receptor.
J Biol Chem. 1999 Oct 8;274(41):29122-9., [PubMed]

Sentences

No. Mutations Sentence Comment

65 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Deletion of Glycosylation Sites-Candidate N-linked glycosylation sites at Asn10 and/or Asn1050 were mutated to create proteins lacking one (SUR-N10Q and SUR-N1050Q) or both (SUR-N10Q,N1050Q) putative glycosylation sites. Login to comment 69 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln SUR1, SUR-N10Q, SUR-N1050Q, and SUR-N10Q,N1050Q (20-30 ␮g/100-mm plate) were cotransfected with Kir 6.2 (1-4 ␮g/ 100-mm plate) using pFx-2 (Invitrogen). Login to comment 118 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln Full-length SUR1, SUR-N10Q, SUR-N1050Q, and SUR-N10Q,N1050Q were coexpressed with Kir 6.2 in COS-1 cells and identified by immunoblotting with an antibody to a carboxyl-terminal V5 epitope tag. Login to comment 123 ABCC8 p.Asn10Gln ABCC8 p.Asn10Gln To determine which residues were glycosylated, the predicted glycosylation acceptor sites Asn10 and Asn1050 were mutated either individually (SUR-N10Q, and SUR-N1050Q) or together (SUR-N10Q,1050Q). Login to comment 124 ABCC8 p.Asn10Gln Expression of SUR-N10Q (Fig. 2, third and fourth lanes) and SUR-N1050Q (Fig. 2, fifth and sixth lanes) demonstrated that elimination of either of the Asn10 or Asn1050 glycosylation consensus sites resulted in a significant decrease of mature glycosylated receptor compared with wild type SUR1. Login to comment 125 ABCC8 p.Asn10Gln Furthermore, simultaneous removal of both the Asn10 and Asn1050 sites in SUR-N10Q,N1050Q resulted only in a band that comigrated with the unglycosylated form of the protein (Fig. 2, seventh and eighth lanes), indicating that both Asn10 and Asn1050 were glycosylated in wild type SUR1. Login to comment 126 ABCC8 p.Asn10Gln A reduction in glycosylation on SUR-N10Q relative to wild type SUR1 is in agreement with previous studies suggesting that Asn10 on the SUR1 amino terminus is glycosylated (14, 15). Login to comment 144 ABCC8 p.Asn10Gln Mutation of that site in SUR285PL-N10Q produced a single band at the same apparent molecular mass as the unglycosylated SUR285PL protein, and its mobility was not shifted by PNGase F treatment (Fig. 4, third and fourth lanes), showing that Asn10 is glycosylated in the SUR285PL fusion protein. Login to comment 156 ABCC8 p.Asn10Gln Glycosylation of SUR285PL and SUR285PL-N10Q fusion proteins. Login to comment 157 ABCC8 p.Asn10Gln Autoradiograms are shown of SUR285PL and SUR285PL-N10Q fusion proteins that were translated in vitro in the presence of [35 S]Met and canine pancreatic microsomes. Login to comment 158 ABCC8 p.Asn10Gln Asn10 in SUR285PL-N10Q was mutated to remove the N-glycosylation acceptor site. Login to comment