ABSTRACT Intracellular malaria parasites export many proteins into their host cell, inserting several into the erythrocyte plasma membrane to enable interactions with their external environment. While static techniques have identified some surface-exposed proteins, other candidates have eluded definitive localization and membrane topology determination. Moreover, both export kinetics and the mechanisms of membrane insertion remain largely unexplored. We introduce Reporter of Insertion and Surface Exposure (RISE), a method for continuous nondestructive tracking of antigen exposure on infected cells. RISE utilizes a small 11-amino acid (aa) HiBit fragment of NanoLuc inserted into a target protein and detects surface exposure through high-affinity complementation to produce luminescence. We tracked the export and surface exposure of CLAG3, a parasite protein linked to nutrient uptake, throughout the Plasmodium falciparum cycle in human erythrocytes. Our approach revealed key determinants of trafficking and surface exposure. Removal of a C-terminal transmembrane domain aborted export. Unexpectedly, certain increases in the exposed reporter size improved the luminescence signal, but other changes abolished the surface signal, revealing that both size and charge of the extracellular epitope influence membrane insertion. Marked cell-to-cell variation with larger inserts containing multiple HiBit epitopes suggests complex regulation of CLAG3 insertion at the host membrane. Quantitative, continuous tracking of CLAG3 surface exposure thus reveals multiple factors that determine this protein’s trafficking and insertion at the host erythrocyte membrane. The RISE assay will enable study of surface antigens from divergent intracellular pathogens. IMPORTANCE Malaria parasites invade and replicate within red blood cells of their human or animal hosts to avoid immune detection. At the same time, these parasites insert their own proteins into the host membrane to scavenge plasma nutrients, facilitate immune evasion, and perform other essential activities. As there is broad interest in developing vaccines and antimalarial therapies against these surface-exposed antigens, robust methods are needed to examine how and when parasite proteins insert at the host membrane. We therefore developed and used Reporter of Insertion and Surface Exposure (RISE) to track parasite antigen exposure. Using RISE, we followed the time course of membrane insertion for CLAG3, a conserved protein linked to a nutrient uptake channel on infected erythrocytes. We found that CLAG3 insertion occurs at specific parasite stages and that this insertion is required for the formation of the nutrient uptake channel. We also varied the size and charge of the extracellular domain to define constraints on protein insertion at the host membrane. Single-cell imaging revealed that some cells continued to export CLAG3 even with large extracellular loops, suggesting sophisticated strategies used by malaria parasites to control their interactions with host plasma.
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