Examining Rhodopsin Folding and Assembly through Expression of Polypeptide Fragments

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.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). These chaperones also display a prolonged interaction with misfolded and aggregated proteins, leading to their retention in the ER (5-7). Nascent polypeptides are targeted to the ER membrane by a signal recognition particle-dependent mechanism. The subsequent translocation of the polypeptide into the ER lumen and the integration of transmembrane segments into the ER membrane are mediated by a protein channel that forms the translocon (8). Calnexin is part of a multimeric protein complex, including calreticulin, BiP, GRP94, and Erp57, that "proofreads" the protein folding process in the ER (7, 9 -11).N-Glycosylation is a co-translational event involving the en bloc transfer of a Glc 3 Man 9 GlcNAc 2 oligosaccharide from a dolichol donor to a lumenal acceptor site (Asn-X-Ser/Thr) in the nascent chain by the ER oligosaccharyl transferase (12-14). Co-translational removal of the terminal glucose residue occurs rapidly by the action of ␣-glucosidase I, followed by the successive removal of the second and third glucose residues by ␣-glucosidase II (15-19). The monoglucosylated oligosaccharide can be regenerated by a glucosyltransferase that acts posttranslationally on unfolded proteins (20). Calnexin and calreticulin are lectins that interact with the Glc 1 Man 9 -5 GlcNAc 2 oligosaccharide (21-26), and calnexin-bound monoglucosyl glycoproteins are protected from ␣-glucosidase II removal of ...

Section: N-glycosylation-dependent Interaction Of Calnexin Withmentioning

confidence: 75%

Calnexin Interaction with N-Glycosylation Mutants of a Polytopic Membrane Glycoprotein, the Human Erythrocyte Anion Exchanger 1 (Band 3)

Popov¹,

Reithmeier²

1999

“…DISCUSSION Few attempts have been made to prepare and characterize peptides corresponding to more than one domain of a GPCR. Notable exceptions are the reconstitution studies on parts of bacteriorhodopsin, rhodopsin, and Ste2p (27)(28)(29). Although these studies expressed, and in some cases isolated, long pieces of these heptahelical proteins, no detailed biophysical analyses were conducted.…”

Section: Analysis Of Peptidesmentioning

confidence: 99%

Synthesis and Biophysical Characterization of a Multidomain Peptide from a Saccharomyces cerevisiae G Protein-coupled Receptor

Naider

Ding

VerBerkmoes

et al. 2003

We attached peptides corresponding to the seventh transmembrane domain (TMD7) of the ␣-mating factor receptor (Ste2p) of Saccharomyces cerevisiae to a hydrophilic, 40-residue fragment of the carboxyl terminus of this G protein-coupled receptor. Peptides corresponding to (a) the 40-residue portion of the carboxyl tail (T-40), (b) the tail plus a part of TMD7 (M7-12-T40), and (c) to the tail plus the full TMD7 (M7-24-T40) were chemically synthesized and purified. The molecular mass and primary sequence of these peptides were confirmed by mass spectrometry and tandem mass spectrometry procedures. Circular dichroism (CD) revealed that T-40 was disordered in phosphate buffer and in the presence of 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-racemic-(1-glycerol)] bilayers. In contrast, M7-12-T40 and M7-24-T40 peptides were partially helical in the presence of vesicles, and difference CD spectroscopy showed that the transmembrane regions of these peptides were 42 and 94% helical, respectively. CD analysis also demonstrated that M7-24-T40 retained its secondary structure in the presence of 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-racemic-(1-glycerol)] micelles at 0.5 mM concentration. Thus, the tail and the transmembrane domain of the multidomain 64-amino acid residue peptide manifest individual conformational preferences. Measurement of tryptophan fluorescence indicated that the transmembrane domain integrated into bilayers in a manner similar to that expected for this region in the native state of the receptor. This study demonstrated that the tail of Ste2p can be used as a hydrophilic template to study transmembrane domain structure using techniques such as CD and NMR spectroscopy.The determination of the structure of G protein-coupled receptors remains an unfulfilled goal of structural biologists with the notable exception of the solution of a high resolution structure of rhodopsin by x-ray crystallography (1). These membrane-bound, information-transducing proteins are acknowledged as a major family of proteins with thousands of representatives encoded by human genes (2, 3). Moreover, the impressive successes in the development of pharmaceuticals targeting G protein-coupled receptors (GPCRs) 1 have created much interest in their structure and mechanism of action (4). Because of the difficulty in obtaining refraction grade crystals of most membrane proteins and their large size, neither x-ray diffraction nor NMR approaches are readily applied to intact receptors. As a route to gain information relevant to these and other membrane proteins such as transporters, many laboratories have taken a reductionist approach and are examining relatively short peptides corresponding to loops and single transmembrane domains (TMDs; Refs. 5-7). A problem encountered in these studies is the poor solubility exhibited by TMDs that leads to difficulty in purifying and conducting biophysical investigations on these molecules. Moreover, although important insights into the conformational preference...

“…Besides the heptahelical membrane proteins BR (7)(8)(9)(10)(11)27), rhodopsin (28,29), adrenergic receptor (30), and muscarinic acetylcholine receptor (31), such studies also include members of other protein families that form helical bundle structures, like the voltagegated sodium channel (32), yeast a-factor transporter (33), or lactose permease (34). Specifically, in the case of BR it has been shown that proteolytic fragments comprising the two helices AB 2 or FG reassociate with the complementary five-helix fragments CG or AE, respectively, to form the native chromophore (7)(8)(9)27).…”

Section: Bacteriorhodopsin (Br)mentioning

confidence: 99%

Refolding of Bacteriorhodopsin from Expressed Polypeptide Fragments

Marti

1998

115

112

Bacteriorhodopsin is a heptahelical membrane protein that can be refolded to the native state following denaturation. To analyze the in vitro folding process with independent structural domains, eight fragments comprising two (AB, FG), three (AC, EG), four (AD, DG) or five (AE, CG) of the transmembrane segments were produced by expression in Escherichia coli. The polypeptides were purified to homogeneity by solvent extraction of E. coli membranes, repeated phase separation, and anion-exchange chromatography employing the C-terminal tail of bacteriorhodopsin for adsorption. Upon reconstitution into phospholipid/detergent micelles pairs of complementary fragments (AB⅐CG, AC⅐DG, AD⅐EG, and AE⅐FG) assembled in the presence of retinal to regenerate the characteristic bacteriorhodopsin chromophore with high efficiency. Together with previous studies, these results demonstrate that the covalent connections in each of the six interhelical loops are dispensable for a correct association of the helices. The different loops, however, contribute to the stability of the folded structure, as shown by increased susceptibilities toward denaturation in SDS and at acidic pH, and decreased Schiff base pK a values for the AB⅐CG, AC⅐DG, AD⅐EG, and AE⅐FG complexes, compared with the intact protein. Notably, the heptahelical bundle structure was also generated by all possible combinations of pairs of overlapping fragments, containing one (AC⅐CG, AD⅐DG, AE⅐EG), two (AD⅐CG, AE⅐DG), or three (AE⅐CG) redundant helices. The spectral properties of the chromophores indicate that the retinal-binding pocket of the AC⅐CG, AD⅐CG, and AE⅐CG complexes is formed by helices A and B of the respective N-terminal fragment and the C-terminal CG fragment, whereas the AD⅐DG, AE⅐DG, and AE⅐EG complexes are likely to adopt a heptahelical bundle structure analogous to AD⅐EG. The combined data show that the specificity of the helix assembly of bacteriorhodopsin is influenced by connectivities provided by the C-D and E-F surface loops. Bacteriorhodopsin (BR)1 is an integral membrane receptor that functions as a light-driven proton pump in the purple membrane of Halobacterium salinarium (1-4). Several features make BR highly attractive for in vitro studies of membrane protein folding and assembly. First, the denatured apoprotein can be spontaneously refolded to the native state with quantitative recovery of secondary structure, chromophore binding, and proton-pumping activity (5, 6). Reconstitution of the native structure has also been accomplished with complementary combinations of proteolytic fragments and synthetic peptides, comprising one or more of the transmembrane regions (7-11). Furthermore, refolding and chromophore binding has been demonstrated for numerous mutants containing amino acid substitutions, deletions, or insertions (12-15). By using time-resolved spectroscopy, transient intermediates in the folding process of native BR have recently been identified (16, 17). Second, the structure of BR has been solved at high resolution, revealing the detai...