Abstract
The interaction of the endoplasmic reticulum chaperone calnexin with N-glycosylation mutants of a polytopic membrane glycoprotein, the human erythrocyte anion exchanger (AE1), was characterized by cell-free translation and in transfected HEK293 cells, followed by co-immunoprecipitation using anti-calnexin antibody. AE1 contains 12-14 transmembrane segments and has a single site of N-glycosylation at Asn-642 in the fourth extracytosolic loop. This site was mutated (N642D) to create a nonglycosylated protein. Calnexin showed a preferential interaction with N-glycosylated AE1 relative to nonglycosylated AE1 both in vitro and in vivo. This interaction could be blocked by inhibition of glucosidases I and II with castanospermine. Calnexin had access to novel N-glycosylated sites created in other extracytosolic loops in AE1 by site-directed or insertional mutagenesis. The interaction with AE1 was enhanced when multiple sites were introduced into the same loop or into two different loops. An association of calnexin with truncated versions of N-glycosylated AE1 was detected after release of the nascent chains from ribosomes with puromycin. The results show that the interaction of calnexin with the polytopic membrane glycoprotein AE1 was dependent on the presence but not the location of the oligosaccharide. Furthermore, calnexin was associated with AE1 after release of AE1 from the translocation machinery.
Highlights
Calnexin, an integral membrane protein, and its soluble homolog, calreticulin, are endoplasmic reticulum (ER)1 chaperones that bind transiently to newly synthesized glycoproteins, thereby promoting their folding and oligomerization (1– 4)
Calnexin Interaction with Polytopic Membrane Glycoproteins are N-glycosylated on a single EC loop, greater than 30 residues in size (47), these proteins are likely to interact with calnexin
In a polytopic membrane protein, various EC domains may not be accessible to interact with calnexin
Summary
Materials—The following is a list of products and their sources: ModulisTM in vitro transcription system and canine pancreatic microsomes (MBI Fermentas), Flexi rabbit reticulocyte lysate and RNasin (Promega), [35S]Met and [␣-35S]ATP (NEN Life Science Products), TransformerTM mutagenesis kit (CLONTECH), T7 sequencing kit (Amersham Pharmacia Biotech), digitonin and Triton X-100 (Roche Molecular Biochemicals), Brij 96 (Sigma), dropping bottle with 20 –30 m filter unit (Wheaton). The endogenous N-glycosylation site at Asn-642 was mutated to Asp (N642D) in the membrane domain construct of AE1-AE1md (residues 386 –911), as described (51). Insertion of EC Loop 4 into EC Loops 1, 2, and 7—Short EC loops in TM were expanded by insertion of a 35-amino acid sequence corresponding to residues 626 – 659 of EC loop 4, containing the endogenous or mutated (N642D) N-glycosylation acceptor site, as described (49, 52). For co-translational interaction, the cDNA corresponding to the AE1md was linearized within the coding region at amino acid positions 696 (NcoI), 714 (NotI), 754 (XbaI), 785 (XbaI), 820 (XbaI), and 854 (NaeI, the endogenous unique restriction site). Beads were washed, followed by incubation with 1% SDS, 5 mM EDTA at 30 °C for 30 min, and diluted with 10 volumes of PBS buffer containing 1% Triton X-100. The fraction of protein that co-immunoprecipitated with calnexin was estimated by determining the intensity of the band interacting with calnexin in the immunoprecipitation fraction divided by the total protein integrated into the microsomes
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