Arenavirus Stable Signal Peptide Is the Keystone Subunit for Glycoprotein Complex Organization

Bederka, Lydia H.; Bonhomme, Cyrille J.; Ling, Emily L.; Buchmeier, Michael J.

Blocking the SSP C-terminal region interferes with plasma membrane trafficking. The signal peptide is required for GPC cleavage, a prerequisite for glycoprotein complex localization at the plasma membrane. We compared glycoprotein expression to phalloidin-defined cell membrane boundaries in the presence or absence of detergent permeabilization using confocal microscopy and a confirmation-specific GP1 antibody (39) . In addition, we used the ER chaperone protein Grp78 to determine intracellular glycoprotein complex localization. Wild-type glycoprotein produced a plasma membrane signal with pseudopod-like membrane exvaginations with moderate intracellular Grp8 staining (Fig. 3A) . A similar phenotype was produced with in trans coexpression of wild-type SSP and GP1/2 and with HA SSP GPC. Further, the HA SSP GPC allowed for SSP detection at the plasma membrane both with and without detergent treatment. In the absence of detergent permeabilization, the GP1/2 precursor lacking its SSP resulted in the lack of plasma membrane expression and a "leaky" ER staining, which was confirmed by Grp78 staining. Similarly, cotransfection of the SSP-HA and GP1/2 failed to yield plasma membrane detection of the GP complex. Rather, the entire complex was retained within the ER. Taking into account the defect in glycopro-tein processing observed in transfected cell lysates (Fig. 2) , blocking the SSP C-terminus interferes with the ability of the GP complex to traffic from the ER to the Golgi for processing by the cellular SKI-1/S1P enzyme to produce mature GP subunits (13, 14) .