THE FUNCTION OF A NOVEL PLASMODIUM ER PROTEIN IN EFFECTOR PROTEIN EXPORT - 7. PROJECT SUMMARY MALARIA CONTINUES TO BE ONE OF THE DEADLIEST PARASITE-BORNE DISEASES IN THE WORLD. RESISTANCE TO ANTIMALARIALS IN ENDEMIC REGIONS HAS HIGHLIGHTED THE IMPORTANCE OF THE DEVELOPMENT OF NEW THERAPEUTIC STRATEGIES TO COMBAT THE DEADLY DISEASE. PLASMODIUM FALCIPARUM, THE CAUSATIVE AGENT OF THE MOST SEVERE FORM OF MALARIA, EXPORTS HUNDREDS OF EFFECTOR PROTEINS INTO THE ERYTHROCYTE HOST CELL TO CREATE AN INTRACELLULAR NICHE FOR REPLICATION. THESE EXPORTED EFFECTORS ARE FIRST SENT THROUGH THE SECRETORY PATHWAY TO THE PARASITE VACUOLE (PV) PRIOR TO BEING TRANSLOCATED TO THE RED BLOOD CELL HOST BY THE PLASMODIUM TRANSLOCON OF EXPORTED PROTEINS (PTEX). PRIOR TO EXPORT, EFFECTOR PROTEINS ENTER THE PARASITE ENDOPLASMIC RETICULUM (ER) VIA A SIGNAL PEPTIDE OR AN INTERNAL HYDROPHOBIC DOMAIN. MOST EXPORTED EFFECTORS UNDERGO A PROTEOLYTIC PROCESSING EVENT AS THEY ENTER THE ER WHICH IS CARRIED OUT BY EITHER PLASMEPSIN V OR SIGNAL PEPTIDASE AND YIELDS AN EXPORT-COMPETENT MATURE N- TERMINUS. WHILE THE MATURE N-TERMINUS IS IMPORTANT FOR TRANSLOCATION ACROSS THE PV MEMBRANE, IT IS UNCLEAR HOW EXPORTED PROTEINS ARE IDENTIFIED BY PTEX OR WHETHER SPECIALIZED MACHINERY GOVERNS THEIR TRAFFICKING THROUGH THE SECRETORY PATHWAY. INTERESTINGLY, THE PTEX UNFOLDASE HSP101 SHOWS A UNIQUE DUAL LOCALIZATION TO BOTH THE ER AND PV, SUGGESTING CARGO RECOGNITION MAY BEGIN UPSTREAM OF THE VACUOLE. WE USED COMPARATIVE PROXIMITY LABELING TO SEARCH FOR HSP101-INTERACTING PROTEINS INVOLVED IN EARLY EXPORT PATHWAY EVENTS AND IDENTIFIED PF3D7_0625400, A NOVEL, PLASMODIUM-SPECIFIC ER PROTEIN UNIQUELY ENRICHED USING HSP101 AS BAIT. IMPORTANTLY, KNOCKDOWN OF PF3D7_0625400 WAS LETHAL AND RESULTED IN A GENERAL BLOCK IN PROTEIN EXPORT BUT DID NOT IMPACT TRAFFICKING OF PTEX COMPONENTS, SUGGESTING A SPECIFIC ROLE IN THE EXPORT PATHWAY. REMARKABLY, DEPLETION OF PF3D7_0625400 RESULTED IN DIFFERENTIAL TRAFFICKING DEFECTS WITH SIGNAL PEPTIDE-CONTAINING EXPORTED PROTEINS ACCUMULATING IN THE PV WHILE EXPORTED PROTEINS THAT LACK A SIGNAL PEPTIDE AND ENTER THE SECRETORY PATHWAY VIA AN INTERNAL HYDROPHOBIC DOMAIN WERE ARRESTED IN THE ER. WE HYPOTHESIZE THAT NVEL IS PART OF A SPECIALIZED ER CHAPERONE SYSTEM DEDICATED TO PROTEIN EXPORT THAT ENABLES PTEX CARGO RECOGNITION BY HSP101 EARLY IN THE SECRETORY PATHWAY. THIS PROPOSAL WILL REVEAL EVENTS IN THE ER REQUIRED FOR EXPORTED CARGO SELECTION AND OPEN THE DOOR TO DECIPHERING THE EARLIEST STEPS IN EFFECTOR PROTEIN TRAFFICKING. MOREOVER, THIS PROPOSAL ALSO CHARACTERIZES A POTENTIAL TARGET FOR THE DEVELOPMENT OF NEW THERAPEUTIC STRATEGIES AGAINST MALARIA INFECTION. AIM 1 WILL EXPLORE PF3D7_0625400 AS PART OF A CHAPERONE SYSTEM DEDICATED TO A SUBSET OF EXPORT PROTEINS THAT CONTAIN SPECIFIC BIOCHEMICAL FEATURES FOR ENTRY. AIM 2 WILL CHARACTERIZE THE NVEL-HSP101 INTERACTION AND DETERMINE ITS IMPORTANCE FOR PROTEIN EXPORT.