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

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

Mutations within the FLLL motif inhibit glycoprotein trafficking. The FLLL glycoprotein mutants that allowed GPC processing were examined for their ability to traffic to the plasma membrane using confocal microscopy. We used colocalization with the Golgi protein mannosidase II (MannII) as an indicator for glycoprotein exit from the ER. Wild-type GPC and the reconstituted wild-type GPC (SSP and GP1/2) allowed for GP1 localization with MannII as well as an extensive, defined plasma membrane (Fig. 4C ). The GP1/2 protein, lacking the native SSP, produced less localization with the Golgi marker, showed extensive cytoplasmic accumulation of the precursor protein akin to retention within the ER, and failed to express surface GP1 above mock transfection levels via flow cytometry (Fig. 4D ). The FALA GPC and the YALL GPC produced extensive cytoplasmic staining, indicating ER accumulation, as well as Golgi colocalization (Fig. 4C) . While the FALA GPC and the YALL GPC generated cleaved GP2 and Golgi marker colocalization, these mutant glycoproteins produced a greater-than-2-fold reduction in plasma membrane localization compared to that of the WT GPC and the GP1/2 and WT SSP levels (Fig. 4D) . The microscopy results strengthen the importance of an intact FLLL motif so SSP may orchestrate glycoprotein maturation processes downstream of targeting the nascent protein to the secretory pathway.