Peptide-Based Interactions with Calnexin Target Misassembled Membrane Proteins into Endoplasmic Reticulum-Derived Multilamellar Bodies

https://doi.org/10.1016/j.jmb.2008.02.056Get rights and content

Abstract

Oligomeric assembly of neurotransmitter transporters is a prerequisite for their export from the endoplasmic reticulum (ER) and their subsequent delivery to the neuronal synapse. We previously identified mutations, e.g., in the γ-aminobutyric acid (GABA) transporter-1 (GAT1), which disrupted assembly and caused retention of the transporter in the ER. Using one representative mutant, GAT1-E101D, we showed here that ER retention was due to association of the transporter with the ER chaperone calnexin: interaction with calnexin led to accumulation of GAT1 in concentric bodies corresponding to previously described multilamellar ER-derived structures. The transmembrane domain of calnexin was necessary and sufficient to direct the protein into these concentric bodies. Both yellow fluorescent protein-tagged versions of wild-type GAT1 and of the GAT1-E101D mutant remained in disperse (i.e., non-aggregated) form in these concentric bodies, because fluorescence recovered rapidly (t1/2 500 ms) upon photobleaching. Fluorescence energy resonance transfer microscopy was employed to visualize a tight interaction of GAT1-E101D with calnexin. Recognition by calnexin occurred largely in a glycan-independent manner and, at least in part, at the level of the transmembrane domain. Our findings are consistent with a model in which the transmembrane segment of calnexin participates in chaperoning the inter- and intramolecular arrangement of hydrophobic segment in oligomeric proteins.

Introduction

Neurotransmitters are released into the synaptic cleft from the presynaptic specialization and their action at pre- and postsynaptic receptors is limited in most instances by rapid re-uptake via specific transporters. Neurotransmitter transporters are thus responsible for rapid inactivation of the signals evoked at the postsynaptic neuronal membrane by their cognate neurotransmitters. Prominent among the protein families involved in this process is the neurotransmitter:sodium symporter (NSS) family, which includes carriers for serotonin (SERT), dopamine (DAT), norepinephrine, glycine, γ-amino butyric acid [GABA transporter (GAT)1–4] and a range of amino-acid and orphan transporters.1 The mammalian members of the NSS family have been extensively studied.2 They are predicted to possess 12 transmembrane segments and to share the same topology and several additional structural features based on the sequence similarity among various NSS members. The structure of a homologous bacterial protein, leucine transporter LeuT, has recently been solved by X-ray crystallography3 and thus serves as a template to explore the structural basis of the translocation process.4

Biogenesis of membrane proteins relies on the following initial reactions: (i) the first hydrophobic peptide segment emerges from the ribosome and is (ii) recognized by the signal recognition particle, which mediates translation arrest and (iii) targeting to the translocon complex (Sec61) for insertion of the hydrophobic segment into the endoplasmic reticulum (ER) membrane. Translation resumes and, hence, membrane insertion of the remaining hydrophobic segments is concomitant with protein synthesis. Folding and possible oligomeric assembly occurs in the ER (for detailed overview, see Refs. 5, 6). Membrane proteins are hydrophobic and hence prone to aggregation. To prevent protein aggregation and to direct the folding pathway to a “native” conformation, the newly synthesized proteins appearing in the ER membrane are immediately bound by the ER resident chaperones—BiP, calreticulin, PDI, etc.7 Interactions of ER chaperones with luminal substrate proteins have been extensively characterized. However, for membrane proteins, information is limited. Chaperones that assist folding of soluble proteins are unlikely to suffice because they cannot prevent aggregation of the membrane-embedded protein segments. In many instances, the membrane-embedded portion represents the bulk of the transmembrane (TM) protein.

The number of identified chaperones for membrane proteins is very limited. The subunits of the translocon complex (SecYEG in prokaryotes, Sec61 in eukaryotic organisms) help membrane proteins to assemble.8 Similarly, a bacterial YidC, which mediates Sec-independent protein insertion into the membrane, assists transmembrane domain folding.9 A membrane protein chaperone, Shr3p, has been identified in yeast.10, 11 Calnexin is an ER resident membrane-associated chaperone with a type I membrane topology: it has an N-terminal luminal lectin domain, a transmembrane domain and a cytosolic C-terminus rich in acidic residues. The sequence of calnexin is conserved in vertebrates,12 and related proteins have been identified in all eukaryotes, including yeast.13 The fundamental function of calnexin is to bind the carbohydrate moieties attached to the newly synthesized proteins in the ER and to assist the folding of the bound proteins.14, 15 In addition to assisting protein folding, calnexin participates in protein degradation by passing the non-refoldable substrates to ER degradation enhancing mannosidase-like protein.16 Several studies reported the glycan-independent interactions of calnexin with its substrates, including interactions mediated by its TM region.17, 18 Calnexin has been implicated in several genetic diseases, including cystic fibrosis due to retention of cystic fibrosis transmembrane conductance regulator (CFTR),19 emphysema resulting from α1-antitrypsin deficiency20 and hemophilia caused by misfolding of clotting factor VIII.21 It has been shown that calnexin may segregate into specialized compartments in the ER.22, 23 Moreover, overexpression of calnexin was shown to lead to formation of multilamellar bodies that retain misfolded CFTR molecules.24

Our earlier work identified mutations within the second transmembrane domain of GAT1, which impaired the ability of the protein to form oligomers and to traffic to the cell surface25: the E101D mutant of GAT1 translocates substrate in a manner indistinguishable from that of the wild-type transporter and is thus likely to adopt a native conformation. Nevertheless, GAT1-E101D is not efficiently exported from the ER because it fails to assemble correctly. The available biochemical or structural data cannot explain why the oligomerization and ER export of the mutant GAT1 fail. Here, we show that calnexin actively participates in the retention of the misassembled GAT1 molecules in the ER. GAT1 and its E101D mutant are retained by interaction with calnexin in concentric bodies previously referred to as organized smooth ER (OSER) structures.26 Furthermore, the interactions between GAT1 molecules and calnexin are only partially abolished by inhibition of glycan recognition. Recognition of the E101D GAT1 assembly defect by calnexin is peptide-based and occurs, at least in part, at the transmembrane domain level.

Section snippets

Modulation of ER calcium and inhibition of glucosidases partially rescues misassembled GAT1 mutant at the cell surface

Membrane proteins that fail to fold correctly in the ER are known to associate with various ER resident chaperones, such as BiP, calreticulin or calnexin.7 Treatment with agents that modulate Ca2+ in the ER by inhibition of the sarcoplasmic endoreticular Ca2+ ATPase in some instances rescued the misfolded ΔF508-CFTR mutants and allowed for its export to the cell surface.27, 28 We tested whether alteration of Ca2+ homeostasis in the ER afforded the rescue of cell surface targeting of the

Discussion

To the best of our knowledge, we demonstrate for the first time that endogenous calnexin is targeted to the multilamellar OSER membrane compartment. We consistently observed these structures regardless of whether cells were transfected or endogenous calnexin was visualized by immunostaining. Thus, these structures cannot be accounted for by experimental artifacts resulting from either the use of a fluorescently tagged protein or fixation and permeabilization, which is inherent in staining with

Plasmids and cDNA constructs

The cDNA encoding calnexin was isolated by PCR from a library generated from the total RNA of HEK293 cells using RT-PCR and was cloned into pEYFP vector (Clontech) using SacI/AccI restriction sites. Truncations of calnexin were generated by PCR. Rabbit anti-calnexin antibody was from Stressgen (SPA-860). Plasmid encoding GFP-tagged PS1-dn was kindly provided by Christian Haass (Munich, Germany). Plasmid encoding mGluR1-CFP was kindly provided by Laurent Fagni (Montpellier, France). Secondary

Acknowledgements

We thank Marion Holy for superb maintenance of cell culture. We are also grateful to Yvonne Vallis (Cambridge, UK) for providing rat embryo fibroblasts. This work was supported by Austrian Science Fund/FWF project programme grant SFB-35, Austrian National Bank grant 10507 (to H.F.) and Austrian Science Foundation grants P18072 (to H.F.), P15034 (to M.F.) and P17076 (to H.H.S.). V.M.K. is supported by a FEBS Long-term Fellowship at the MRC Laboratory of Molecular Biology.

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