Analysis of the transmembrane domain of influenza virus neuraminidase, a type II transmembrane glycoprotein, for apical sorting and raft association

Abstract

Influenza virus neuraminidase (NA), a type II transmembrane protein, is directly transported to the apical plasma membrane in polarized MDCK cells. Previously, it was shown that the transmembrane domain (TMD) of NA provides a determinant(s) for apical sorting and raft association (A. Kundu, R. T. Avalos, C. M. Sanderson, and D. P. Nayak, J. Virol. 70:6508-6515, 1996). In this report, we have analyzed the sequences in the NA TMD involved in apical transport and raft association by making chimeric TMDs from NA and human transferring receptor (TR) TMDs and by mutating the NA TMD sequences. Our results show that the COOH-terminal half of the NA TMD (amino acids [aa] 19 to 35) was significantly involved in raft association, as determined by Triton X-100 (TX-100) resistance. However, in addition, the highly conserved residues at the extreme NH(2) terminus of the NA TMD were also critical for TX-100 resistance. On the other hand, 19 residues (aa 9 to 27) at the NH(2) terminus of the NA TMD were sufficient for apical sorting. Amino acid residues 14 to 18 and 27 to 31 had the least effect on apical transport, whereas mutations in the amino acid residues 11 to 13, 23 to 26, and 32 to 35 resulted in altered polarity for the mutant proteins. These results indicated that multiple regions in the NA TMD were involved in apical transport. Furthermore, these results support the idea that the signals for apical sorting and raft association, although residing in the NA TMD, are not identical and vary independently and that the NA TMD also possesses an apical determinant(s) which can interact with apical sorting machineries outside the lipid raft.

FIG. 1

Constructs of chimeric and mutant NA TMDs. Portions of the TR and NA TMDs were swapped to construct four chimeric TMDs (construct no. 5 to 8), as described in Materials and Methods. For the mutants (construct no. 9 to 15), sets of 4 or 5 amino acids in the NA TMD were replaced by alanines. The six amino acids at the NH₂ terminus of TRΔ57 and the other constructs consist of the first two residues (MM) plus the last four residues (KPKR) of the TR cytoplasmic tail. □, amino acid from NA sequence; ○, amino acid from the TR sequence. The lowercase letters represent amino acids neither from the NA sequence nor from the TR sequence but introduced or replaced due to creation of restriction enzyme sites. The numbers at the top represent amino acid positions with respect to the NA sequence.

FIG. 2

Expression of different chimeric and mutant proteins in stable MDCK cells. Stable, transfected MDCK cells were established and cultured for 2 days and induced for 16 h with 2 μM of CdCl₂. Cells were then pulse-labeled with 150 μCi of [³⁵S]-Easy Tag Express protein-labeling mix for 1 h, followed by a 2-h chase (except for construct no. 10, which was chased for 6 h). Cells were then lysed, and the lysates were immunoprecipitated, analyzed by SDS-PAGE, and autoradiographed. Lane M, standard protein marker; lanes 1 to 15, constructs 1 to 15, respectively (numbering of constructs is shown in Fig. 1). The numbers on the righthand side represent the following: 1, nonspecific band; 2, mature protein band; 3, immature protein band (construct no. 2 to 15). Note that there is some migration variation among the different chimeric and mutant proteins.

FIG. 3

Polarized cell surface distribution (A) and TX-100 extraction (B) of chimeric NA TMD proteins. For panel A, confluent monolayers of MDCK cell lines were grown on filters for 3 to 4 days, induced for 16 h with 2 μM CdCl₂, pulse-labeled for 2 h with 300 μCi of [³⁵S]-Easy Tag Express protein-labeling mix and chased for 2 h. The apical (lanes A) and basolateral (lanes B) surface proteins from parallel cultures were biotinylated, isolated by anti-TR antibodies, analyzed by SDS-PAGE, and autoradiographed. For panel B, cells were grown on 35-mm petri dishes for 2 days and induced with 2 μM of CdCl₂ for 16 h, pulse-labeled with 150 μCi of [³⁵S]-Easy Tag Express protein-labeling mix for 2 h, followed by a 2-h chase. Cells were extracted on ice with extraction buffer containing 1% TX-100 for 10 min, as described in Materials and Methods. TX-100 insoluble (lanes I) and soluble (lanes S) proteins were immunoprecipitated with anti-TR antibodies, analyzed by SDS-PAGE, and autoradiographed.

FIG. 4

Polarized surface distribution (A) and TX-100 extraction (B) of mutant NA TMD proteins. Experimental conditions were as described for Fig. 3, except that the mutant protein NA5A11 was chased for 6 h. Lanes: A, apical surface proteins; B, basolateral surface proteins; I, insoluble proteins; S, soluble proteins.

FIG. 5

Conservation of amino acids in NA TMD. TMD sequences of 37 NA (subtype N1) proteins were compared. For each position, a conservation value was calculated by dividing the percentage of sequences having the most common amino acid by the number of different amino acids found at that position as described by Lin et al. (18).

FIG. 6

The effect of alteration of NA TMD sequences on apical sorting and raft association. The increasing height or depth of boxes indicates the increasing effects of those amino acids.