Background: PI(4,5)P 2 -and tyrosine phosphorylation-dependent unconventional secretion of FGF2 is mediated by direct translocation across the plasma membrane. Results: PI(4,5)P 2 -mediated membrane recruitment causes oligomerization of tyrosine-phosphorylated FGF2 that, in turn, triggers the formation of a lipidic membrane pore. Conclusion: Membrane-inserted FGF2 oligomers represent intermediates of membrane translocation during unconventional secretion. Significance: Mechanistic insight into a novel self-sustained mechanism of protein translocation across membranes is provided.

A key process in the lifecycle of the malaria parasite Plasmodium falciparum is the fast invasion of human erythrocytes. Entry into the host cell requires the apical membrane antigen 1 (AMA-1), a type I transmembrane protein located in the micronemes of the merozoite. Although AMA-1 is evolving into the leading blood-stage malaria vaccine candidate, its precise role in invasion is still unclear. We investigate AMA-1 function using live video microscopy in the absence and presence of an AMA-1 inhibitory peptide. This data reveals a crucial function of AMA-1 during the primary contact period upstream of the entry process at around the time of moving junction formation. We generate a Plasmodium falciparum cell line that expresses a functional GFP-tagged AMA-1. This allows the visualization of the dynamics of AMA-1 in live parasites. We functionally validate the ectopically expressed AMA-1 by establishing a complementation assay based on strain-specific inhibition. This method provides the basis for the functional analysis of essential genes that are refractory to any genetic manipulation. Using the complementation assay, we show that the cytoplasmic domain of AMA-1 is not required for correct trafficking and surface translocation but is essential for AMA-1 function. Although this function can be mimicked by the highly conserved cytoplasmic domains of P. vivax and P. berghei, the exchange with the heterologous domain of the microneme protein EBA-175 or the rhoptry protein Rh2b leads to a loss of function. We identify several residues in the cytoplasmic tail that are essential for AMA-1 function. We validate this data using additional transgenic parasite lines expressing AMA-1 mutants with TY1 epitopes. We show that the cytoplasmic domain of AMA-1 is phosphorylated. Mutational analysis suggests an important role for the phosphorylation in the invasion process, which might translate into novel therapeutic strategies.

Background: Unconventional secretion of FGF2 occurs by direct translocation across plasma membranes. Results: The cytoplasmic domain of ATP1A1 directly interacts with FGF2 and is required for FGF2 secretion. Conclusion: ATP1A1 supports unconventional secretion by recruiting FGF2 to the inner leaflet of plasma membranes. Significance: A new machinery component required for unconventional secretion of FGF2 was identified and validated.

a b s t r a c tInfluenza A Neuraminidase is essential for virus release from the cell surface of host cells. Given differential structures of the N-terminal sequences including the transme mbrane domains of neuraminidase subtypes , we investigated their contribution to transport and localization of subtypes N1, N2 and N8 to the plasma membrane. We generated consensus sequences from all protein entries available for these subtypes. We found that 40N-terminal the forty N-terminal amino acids are sufficient to confer plasma membran e localization of fusion proteins, albeit with different efficiencies. Strikingly, subtle differences in the prima ry structure of the part of the transmemb rane domain that resides in the exoplasmic leaflet of the membrane have a major impact on transport efficiency, providing a potential target for the inhibition of virus release.