Post-translational N-glycosylation of type I transmembrane KCNE1 peptides: implications for membrane protein biogenesis and disease

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.

FIGURE 1.

KCNE1 subunits are co- and post-translationally N-glycosylated. A, representative fluorographs for wild type E1 expressed alone (open circles), in the presence of proteasome inhibitors (MG-132) (diamonds), or expressed with Q1 channel subunits (filled circles). 2-Gly: diglycosylated; 1-Gly: monoglycosylated; 0-Gly: unglycosylated. Cells were pulsed for 2 min in order to observe post-translational N-glycosylation, and chased for the indicated times. The immunoprecipitated E1 proteins were separated by electrophoresis and detected by autoradiography. B, plots of the diglycosylated signal over chase-time. Top: raw signal intensity; bottom: percentage of the diglycosylated isoform with respect to total KCNE1 protein.

FIGURE 2.

Post-translational N-glycosylation of KCNE1 occurs primarily at the N26 sequon. A, schematic representation of E1. The positions of the N-linked glycosylation sites and the transmembrane domain (TM) are denoted. Representative fluorographs of the E1 N-glycosylation mutants expressed alone (B) or with Q1 subunits (C). Cells were pulsed for 2 min to observe post-translational N-glycosylation, and chased for the indicated times. The immunoprecipitated E1 proteins were separated by electrophoresis and detected by autoradiography. N26Q (squares), N5Q (inverted triangles), and T7I (triangles). D, graphs of densitometric analysis of the E1 N-glycosylation mutants expressed alone (open symbols) or with Q1 subunits (filled symbols). The percentage of the maximally glycosylated forms with respect to total protein is plotted for each time point. Data plotted (n = 4–5) are mean ± S.E. for each chase point. After 3 min, the maximally glycosylated forms of N5Q and T7I increased whereas N26Q remained relatively constant. Dotted line segregates the N5 and N26 sequon data.

FIGURE 3.

N-Glycan occupancy effects post-translational N-glycosylation efficiency. A, immunoblots of WT and E1 N-glycosylation mutants from detergent-solubilized cells. 2-Gly: diglycosylated; 1-Gly: monoglycosylated; 0-Gly: unglycosylated; Molecular weight markers are denoted on the left and right. The immaturely (im) and unglycosylated (un) glycoforms were identified by enzymatic deglycosylation (supplemental Fig. S3C). B, bar graph of the percentage of glycosylated WT and mutant E1 subunits. Error bars are S.E. from n = 3–6 immunoblots.

FIGURE 4.

Current properties of KCNQ1 channels co-expressed with KCNE1 N-glycosylation mutants. A, representative families of I_Q1 and I_Ks currents elicited by the pulse protocol shown. The interpulse interval was 30 s. B, representative families of currents recorded from cells expressing Q1 and the E1 N-glycosylation mutants (+N5Q, +T7I, or +N26Q). Arrows mark the rapid activation that is indicative of unpartnered KCNQ1 channels (I_Q1). C, relative mean peak currents (I/I_max) were normalized to the maximal WT I_ks (+E1) and plotted as a function of the pulse voltage (V). Data (n = 3–5) are mean ± S.E.

FIGURE 5.

Compounded hypoglycosylation of KCNE1 reduces cell surface expression via an anterograde trafficking defect. Cell surface labeling of E1 subunits co-expressed with Q1. A, representative immunoblots of cell surface labeling. Lanes denoted as (½ input) are half the sample lysate that was set aside to quantitate the total amount of biotinylated proteins. Beads, lanes represent the cell surface biotinylated proteins that were isolated with streptavidin and separated by SDS-PAGE. The CNX immunoblots were used both to determine the amount of background lysis and to compare the cell surface expression of the mutants to WT. The mature (m), immature (im), and unglycosylated (un) forms were identified by enzymatic deglycosylation (supplemental Fig. S3C). B, quantification of the E1 proteins on the cell surface, which was calculated as described under “Experimental Procedures.” Error bars are S.E. from n = 3–4 immunoblots. C, all glycoforms of E1 equally co-assemble with Q1. Left panel: immunoprecipitation of radiolabeled E1 using antibodies specific for E1, Q1, or non-immune control antibody (−). The ratio of the glycoforms precipitated was the same whether Q1 or E1 antibody was used. Right panel: radiolabeled N-glycosylation mutants were co-immunoprecipitated with α-Q1 antibodies. Co-immunoprecipitations required the presence of Q1 channels, (−)/WT.

FIGURE 6.

Co- and post-translational N-glycosylation of the N26 sequon depends on the hydroxyamino acid. A, left: representative fluorograph of the N5Q+S28T (open hexagons) N-glycosylation mutant expressed alone. The 2 min pulse-chase labeling was performed as denoted in Fig. 2. Right: percentage of the maximally glycosylated forms with respect to total protein at each time point is compared with a predominately co-translational mutant (N26Q line) and post-translational mutant (N5Q line). Data plotted (n = 4) are mean ± S.E. for each chase point. B, cell surface labeling of N5Q+S28T subunits co-expressed with Q1. Left: representative immunoblots of cell surface labeling. Lanes denoted as (½ input) are half the sample lysate that was set aside for quantization. Beads, lanes represent the isolated biotinylated proteins. The CNX immunoblots were used to determine the amount of background lysis. The mature (m), immature (im), and unglycosylated (un) forms were identified as described in supplemental Fig. S3C. Right: quantification of E1 proteins on the cell surface which was calculated as described under “Experimental Procedures.” Error bars are S.E. from n = 3 immunoblots. C, left: representative family of currents from cells co-expressing N5Q+S28T with Q1. Voltage pulse protocol is shown in Fig. 4A. Arrow marks the absence of rapid activation that is indicative of unpartnered Q1 channels (Fig. 4A). Right: comparison of the relative mean peak currents (I/I_max) of the Q1/N5Q mutants with a serine (triangles) or a threonine (hexagons) residue in the N26 sequon (NXS versus NXT).

FIGURE 7.

Model of KCNE1 biogenesis, N-glycosylation, and co-assembly with KCNQ1 channels. N-linked glycans are added to the N5 sequon of E1 subunits during translation (co-translational) and laterally exit the protein translocation channel to integrate into the membrane. Post-translational N-glycosylation of WT subunits at N26 occurs either before (a) or after (b) co-assembly with Q1 channel subunits. Once fully glycosylated, the Q1/E1 complex exits the ER and traffics to the plasma membrane. For the Long QT mutation, T7I, the subunit exits the translocon unglycosylated, and is a poor substrate (compared with WT) for post-translational N-glycosylation. Unglycosylated T7I subunits readily co-assemble with Q1 subunits, resulting in complexes that have an anterograde trafficking defect, which significantly reduces cell surface expression.