Determination of the orientation of an integral membrane protein and sites of glycosylation by oligonucleotide-directed mutagenesis: influenza B virus NB glycoprotein lacks a cleavable signal sequence and has an extracellular NH2-terminal region.

Abstract

The membrane orientation of the NB protein of influenza B virus, a small (Mr,,000) glycoprotein with a single internal hydrophobic domain, was investigated by biochemical and genetic means. Cell fractionation and protein solubility studies indicate NB is an integral membrane protein, and NB has been shown to be a dimer under nonreducing conditions. Treatment of infected-cell surfaces with proteinase K and endoglycosidase F and immunoprecipitation with a site-specific antibody suggests that the 18-amino-acid NH2-terminal region of NB is exposed at the cell surface. Oligonucleotide-directed mutagenesis to eliminate each of the four potential sites of N-linked glycosylation and expression of the mutant NB proteins in eucaryotic cells suggest that the two sites adjacent to the NH2 terminus are glycosylated. This provides further evidence that NB, which lacks a cleavable NH2-terminal signal sequence, has an exposed NH2 terminus at the cell surface.In eucaryotic cells, glycoproteins are either translocated completely across the membrane into the lumen of the endoplasmic reticulum for soluble proteins destined for secretion or they are asymmetrically integrated into distinct cellular membranes. Integral membrane proteins may span the membrane once or more with the NH2 and COOH termini of the polypeptide chain on either side of the bilayer. These proteins are anchored in membranes by a membranespanning domain which is generally found to consist of 20 or more hydrophobic or uncharged amino acids. It has been established that the orientation of an integral membrane protein is absolute and is maintained regardless of the final destination of the protein (e.g., in the plasma membrane) (reviewed in references 3 and 54). Similarly, the addition of asparagine N-linked carbohydrate to an integral membrane protein is also asymmetric and occurs in the lumen of endoplasmic reticulum vesicles at the amino acid sequence Asn-X-Ser/Thr (reviewed in reference 17). However, determination of the transmembrane orientation and location of the precise sites used for addition of carbohydrate by peptide analysis are often difficult.We have been interested in examining properties of a glycoprotein, NB, of influenza B virus which circumstantial evidence indicated was an integral membrane protein. In the course of these studies we developed a method, based on site-specific mutagenesis and expression of mutant cDNAs in eucaryotic cells, that enabled us to determine the orientation of the protein in membranes and the precise sites of carbohydrate addition.Influenza B virus NB glycoprotein (100 amino acids) is translated from a bicistronic mRNA derived from RNA segment 6 that also encodes the neuraminidase (NA) glycoprotein by using overlapping reading frames (28,48). The first AUG codon in the mRNA is used for the initiation of NB and the second AUG codon, separated from the first AUG codon by four nucleotides, is used to initiate NA. NB * Corresponding author.

The NB protein is an integral component of the membrane of influenza B virus

“…Together, the results of these enzymatic treatments indicate that the majority of the NB protein is expressed on the surface of infected cells. Cycloheximide chase experiments which examined changes in the pattern of NB over 12 h following inhibition of protein synthesis by cycloheximide (50 lag/ml), added at, e.g., 9 h p.i., showed that the non-glycosylated 12 kDa form of the protein was stable over this time, contrary to previous reports of the relative instability of NB (Shaw & Chopin, 1984;Williams & Lamb, 1986). The same experiments showed that removal of the carbohydrate side-chains had a half-life of 2-3 h. Deglycosylation thus appears to be due to …”

“…mainly as a 18 kDa band (as shown in Fig. 1 b) which corresponds to the high mannose form, previously described by Williams & Lamb (1986). As infection progressed different glycosylated forms, presumably with heterogeneous polylactosaminoglycan side-chains (Williams & Lamb, 1988), with apparent molecular masses of 22 kDa, 23-28 kDa and 32-55 kDa became more prominent.…”

“…A hydrophobic domain comprising amino acids 19-40 anchors the protein in the membrane with the I8 amino acid Nterminal domain exposed externally and the C-terminal 60 amino acid segment located within the cytoplasm (Williams & Lamb, 1986). Polylactosaminoglycan side-chains are attached to asparagine residues 3 and 7 (Williams & Lamb, 1988).…”

Co- and Posttranslational Translocation Mechanisms Direct Cystic Fibrosis Transmembrane Conductance Regulator N Terminus Transmembrane Assembly

“…However, when TM1 fails to translocate the chain, topogenic information encoded within TM2 provides a second chance for chains to acquire their correct transmembrane orientation. In this case, we propose that TM2 initiates translocation of its own N terminus flanking residues in a manner similar to an N-trans (type I) signal anchor sequence (2,3,14,55). The unusual feature of this process is that TM1 is posttranslationally directed into the translocon channel from a cytosolic orientation where it then terminates translocation and establishes its proper membrane-spanning topology.…”

Transmembrane Peptide NB of Influenza B: A Simulation, Structure, and Conductance Study

“…For NB the number of subunits in the bundle is not known. Purification of NB on gels using nonreducing conditions revealed the formation of disulfide-linked dimers and higher oligomers (2). Molecular modeling studies on bundles of the transmembrane parts of the peptide have been performed in vacuo with four, five, and six segments (12).…”