The Longin Domain Regulates the Steady-State Dynamics of Sec22 in Plasmodium falciparum

The specificity of vesicle-mediated transport is largely regulated by the membrane-specific distribution of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. However, the signals and machineries involved in SNARE protein targeting to the respective intracellular locations are not fully understood. We have identified a Sec22 ortholog in Plasmodium falciparum (PfSec22) that contains an atypical insertion of the Plasmodium export element within the N-terminal longin domain. This Sec22 protein partially associates with membrane structures in the parasitized erythrocytes when expressed under the control of the endogenous promoter element. Our studies indicate that the atypical longin domain contains signals that are required for both endoplasmic reticulum (ER)/Golgi apparatus recycling of PfSec22 and partial export beyond the ER/Golgi apparatus interface. ER exit of PfSec22 is regulated by motifs within the α3 segment of the longin domain, whereas the recycling and export signals require residues within the N-terminal hydrophobic segment. Our data suggest that the longin domain of PfSec22 exhibits major differences from the yeast and mammalian orthologs, perhaps indicative of a novel mechanism for Sec22 trafficking in malaria parasites.Plasmodium falciparum exhibits a complex network of endomembrane organelles that are unique to this obligate intracellular parasite of human erythrocytes. They include parasite-induced tubules and vesicles in the infected host cell and specialized secretory structures collectively known as the apical complex. The asexual blood stages of the parasite develop within a parasitophorous vacuole (PV) and thus are separated from the external milieu by three lipid bilayers: the parasite plasma membrane (PPM), the PV membrane (PVM), and the erythrocyte plasma membrane. To survive inside these terminally differentiated human erythrocytes, P. falciparum remodels the host cell compartment by exporting numerous proteins into the erythrocyte cytoplasm (12, 15, 49, 50, 57). The mechanisms by which both soluble and membrane-bound proteins are transported, first into the PV lumen, followed by translocation across the PVM and transport within the erythrocyte cytosol, are not fully understood (9). A majority of the exported proteins contain bipartite signals that comprise a “recessed” N-terminal signal sequence and a Plasmodium export element/vacuolar translocation sequence (PEXEL/VTS) that is characterized by the consensus sequence RX(L/I)X(D/E/Q). These signals are predicted to facilitate the transport of proteins into the PV (using their recessed, or N-terminal, signal sequences) and translocation across the PVM (using their PEXEL/VTS motifs) (5, 23, 29, 34). However, a subset of the exported proteins lack either one or both signal elements and may require novel targeting motifs for transport beyond the PPM (20, 43). A majority of the proteins enter the parasite secretory system via the endoplasmic reticulum (ER), where they are incorporated into ER-derived vesicles and then transported through the “unstacked” Golgi bodies to their final destinations (45, 48, 55, 56). Membrane-bound vesicular elements have been detected in the infected host cell cytosol, suggesting the existence of an extraparasitic vesicle-mediated transport process in malaria parasites (22, 47, 52). How vesicle targeting is achieved in P. falciparum parasites remains elusive.Vesicle targeting and fusion in eukaryotic cells involves proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) family (25, 41, 42, 44). SNAREs are “tail-anchored” proteins that function by forming complexes that bridge vesicle and target membranes during fusion (6, 7, 24). Distinct sets of SNARE proteins localize to different intracellular transport pathways using processes that are not well understood. Increasing evidence suggests that the N-terminal regions of SNARE proteins contain signals required for their subcellular localization (4, 31, 53). These N-terminal regions include the three-helical Habc bundles of syntaxin SNAREs and the “profilin-like” folds of long VAMPs (vesicle-associated membrane proteins), also known as longin domains (7, 17, 33, 40, 46). The Sec22 gene products in mammals and yeast are longin domain-containing SNAREs that cycle between the ER and Golgi compartments (3, 19, 31, 32). We have identified a Sec22 ortholog in P. falciparum (PfSec22) that contains a PEXEL/VTS sequence insertion between the α2 and α3 segments of the longin domain preceded by a stretch of hydrophobic residues that spans a region between the β5 and α2 segments (2). In this study, we examined the distribution of PfSec22 in P. falciparum-infected erythrocytes and investigated the role of the atypical longin domain in its steady-state localization. Our data show that the P. falciparum ortholog of Sec22 partially associates with noncanonical destinations (tubovesicular network and intraerythrocytic vesicles) in the infected erythrocytes and that the N-terminal longin domain exhibits a dual function, mediating ER-to-Golgi apparatus trafficking, as well as retrieval from the Golgi apparatus.