title: Structural requirements in the membrane-spanning domain of the paramyxovirus HN protein for the formation of a stable tetramer.
abstract: The paramyxovirus hemagglutinin-neuraminidase (HN) is a type II homotetrameric integral membrane glycoprotein composed of a pair of disulfide-linked dimers that are held together by noncovalent bonds. To determine the role of the internal uncleaved signal-anchor (S/A) domain in stable tetramer formation, cDNA-derived HN mutants containing S/A substitutions were expressed in HeLa cells. The assembly into tetramers and ER-to-Golgi transport of the proteins were examined by sucrose gradient sedimentation and by endoglycosidase treatment. A leucine-scanning substitution analysis of the 19-residue S/A identified 2 polar residues (Ser 31 and Tyr 36) in the C-terminal end of the S/A that were important for the formation of a stable tetramer. While Ala, Cys, and Gly could functionally replace Ser 31 in the formation of a stable tetramer, substitution with Leu or Phe resulted in mutants that were detected as disulfide-linked dimers. These results indicate that a small amino acid in position 31, rather than a specific residue per se, is an important assembly requirement in the S/A. In contrast to the size requirement for position 31, the conservative substitution of Tyr 36 with Phe produced an HN mutant that sedimented as a mixture of dimers, tetramers, and higher order oligomers, suggesting that proper assembly requires a Tyr in this position. The S/A mutants that were detected as disulfide-linked dimers showed only a slight reduction in ER-to-Golgi transport (approximately 50% of WT), consistent with the proposal that the S/A substitutions had affected tetramer stability and not the formation of a transport-competent oligomer. These data indicate that there are different structural requirements for two positions in the C-terminal region of the HN S/A for the assembly of a stable tetramer.
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D002499:Centrifugation, Density Gradient; D004279:DNA, Viral; D006056:Golgi Apparatus / Q000378:metabolism; D015707:HN Protein / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010252:Paramyxoviridae / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship
chemical_list: D000596:Amino Acids; D004279:DNA, Viral; D015707:HN Protein; D008565:Membrane Proteins