Bitopic membrane topology of the stable signal peptide in the tripartite Junín virus GP-C envelope glycoprotein complex.
The stable signal peptide (SSP) of the GP-C envelope glycoprotein of the Junín arenavirus plays a critical role in trafficking of the GP-C complex to the cell surface and in its membrane fusion activity. SSP therefore may function on both sides of the lipid membrane. In this study, we have investigated the membrane topology of SSP by confocal microscopy of cells treated with the detergent digitonin to selectively permeabilize the plasma membrane. By using an affinity tag to mark the termini of SSP in the properly assembled GP-C complex, we find that both the N and C termini reside in the cytosol. Thus, SSP adopts a bitopic topology in which the C terminus is translocated from the lumen of the endoplasmic reticulum to the cytoplasm. This model is supported by (i) the presence of two conserved hydrophobic regions in SSP (hphi1 and hphi2) and (ii) our previous demonstration that lysine-33 in the ectodomain loop is essential for pH-dependent membrane fusion. Moreover, we demonstrate that the introduction of a charged side chain or single amino acid deletion in the membrane-spanning hphi2 region significantly diminishes SSP association in the GP-C complex and abolishes membrane fusion activity. Taken together, our results suggest that bitopic membrane insertion of SSP is centrally important in the assembly and function of the tripartite GP-C complex.
Agnihothram
Sudhakar S
SS
Montana Biotechnology Center, The University of Montana, Science Complex, Room 221, Missoula, MT 59812, USA.
York
Joanne
J
Trahey
Meg
M
Nunberg
Jack H
JH
D000595:Amino Acid Sequence; D002459:Cell Fusion; D002462:Cell Membrane / Q000737:chemistry* / Q000821:virology*; D003593:Cytoplasm / Q000737:chemistry*; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D018051:Junin virus / Q000737:chemistry* / Q000502:physiology; D018613:Microscopy, Confocal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000378:metabolism; D019065:Virus Assembly / Q000502:physiology
D003902:Detergents; D021382:Protein Sorting Signals; D014759:Viral Envelope Proteins; D004072:Digitonin