Molecular and Biochemical Parasitology, 46 (1991) 137-148 © 1991 Elsevier Science Publishers B.V. /0166-6851/91/$03.50 ADONIS 016668519100047M MOLBIO 01518 137 The ring-infected erythrocyte surface antigen of Plasmodiumfalciparum associates with spectrin in the erythrocyte membrane Michael Foley 1, Leann Tiller, William H. Sawyer 3 and Robin F. Anders 1 1The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria, Australia; 2Biochemistry Department, University of La Trobe, Bundoora, Victoria, Australia; and 3Biochemistry Department, University of Melbourne, Parkville, Victoria, Australia (Received 3 July 1990; accepted 26 November 1990) The malaria parasite Plasmodiumfalciparum synthesises a protein, RESA, which associates with the membrane of newly invaded erythrocytes. Using spent supernatants from P.falciparum growing in culture as a source of soluble RESA we have developed an assay to examine the characteristics of RESA binding to the erythrocyte membrane in vitro. RESA associated with the Triton X-100 insoluble proteins on the inner face of the host erythrocyte membrane but did not bind to the outer surface of intact erythrocytes. Other proteins present in culture supernatants did not bind to the erythrocyte membrane. RESA was co-sedimented with the ternary complex formed between actin, spectrin and band 4.1 and co-precipitated with spectrin precipitated with anti-spectrin antibodies. The extent of association between RESA and the inner face of the erythrocyte membrane was reduced by the inclusion of excess purified spectrin in the assay. Thus, RESA appears to be associated with spectrin in the erythrocyte membrane skeleton. Key words: Plasmodiumfalciparum; Spectrin; Bindingassay; Ring-infected erythrocyte surface antigen Introduction Various structural and biochemical changes in the erythrocyte membrane occur during asexual de- velopment of the malarial parasite within erythro- cytes [1]. As the parasite matures the shape and de- formability of the host membrane are altered, and even in the less mature ring-stage parasites there is a measurable decrease in the deformability of the erythrocyte membrane [2]. The network of cyto- skeletal proteins underlying the erythrocyte bilayer is responsible for maintaining the shape and de- formability of the erythrocyte membrane [3]. Thus, parasite proteins that interact with this membrane skeleton may be responsible for changes in these Correspondence (present) address: Michael Foley, Institute of Cell, Animal and Population Biology, University of Edin- burgh, King's Buildings, West Mains Road, Edinburgh EH9 3JN, U.K. Abbreviations: RESA, ring-infected erythrocyte surface anti- gen; TX- 100, Triton X- 100; IOVs, inside-outvesicles. membrane characteristics. Several malarial pro- teins are known to be localised on the cytoplasmic face of the erythrocyte membrane [4,5] and such in- teractions may be important in maintaining the pec- uliar knob structures seen in the membrane of erythrocytes infected with mature stages of P.falci- parum. The ring-infected erythrocyte surface antigen (RESA) is a P.falciparum protein that becomes as- sociated with the membrane of newly invaded erythrocytes. After synthesis, RESA is stored in or- ganelles within the mature parasite and is released into the red cell at the time of merozoite invasion. In mature parasites RESA is largely soluble in the anionic detergent Triton X- 100 (TX- 100) but when associated with the membrane of newly invaded erythrocytes it is TX-100-insoluble, presumably due to an interaction with a component of the eryth- rocyte membrane skeleton [6]. Antibodies to RESA inhibited merozoite in- vasion in vitro [7] and immunisation of Aotus mon- keys with recombinant RESA proteins provided