Post-translational N-Glycosylation of Type I Transmembrane KCNE1 Peptides: IMPLICATIONS FOR MEMBRANE PROTEIN BIOGENESIS AND DISEASE

Tuba Bas,Grace Y Gao,Anatoli Lvov,Kshama D Chandrasekhar,Reid Gilmore,William R Kobertz

doi:10.1074/jbc.m111.235168

Tuba Bas, Grace Y Gao + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m111.235168

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Abstract

N-Glycosylation of membrane proteins is critical for their proper folding, co-assembly and subsequent matriculation through the secretory pathway. Here, we examine the kinetics of N-glycan addition to type I transmembrane KCNE1 K(+) channel β-subunits, where point mutations that prevent N-glycosylation at one consensus site give rise to disorders of the cardiac rhythm and congenital deafness. We show that KCNE1 has two distinct N-glycosylation sites: a typical co-translational site and a consensus site ∼20 residues away that unexpectedly acquires N-glycans after protein synthesis (post-translational). Mutations that ablate the co-translational site concomitantly reduce glycosylation at the post-translational site, resulting in unglycosylated KCNE1 subunits that cannot reach the cell surface with their cognate K(+) channel. This long range inhibition is highly specific for post-translational N-glycosylation because mutagenic conversion of the KCNE1 post-translational site into a co-translational site restored both monoglycosylation and anterograde trafficking. These results directly explain how a single point mutation can prevent N-glycan attachment at multiple sites, providing a new biogenic mechanism for human disease.

Highlights

Asparagine-linked (N-linked) glycosylation is a highly conserved protein modification that eukaryotic cells utilize for the proper folding, assembly, and trafficking of membrane and secreted proteins
A mutation that disrupts the sequon at N5 (T7I), gives rise to an inherited autosomal recessive form of Long QT Syndrome (LQTS), a disorder of the cardiac rhythm that is accompanied with neural deafness, Jervell-Lange-Nielsen Syndrome (JLNS) [19]
After the synthesis of the pulse-labeled E1 protein was complete (ϳ3 min chase) [23], the signal intensity of the diglycosylated form continued to increase over time (Fig. 1B, open circles) such that it became the predominant form of E1

Summary

Introduction

Asparagine-linked (N-linked) glycosylation is a highly conserved protein modification that eukaryotic cells utilize for the proper folding, assembly, and trafficking of membrane and secreted proteins. Given the disease linkage between N-glycosylation and KCNE biology, we determined the kinetics and efficiency of N-glycosylation of the two sequons in E1 and the effects of N-glycan occupancy on co-assembly with Kϩ channel subunits and cell surface expression.

Results

Conclusion