6696–6707 Nucleic Acids Research, 2006, Vol. 34, No. 22 doi:10.1093/nar/gkl942 Published online 5 December 2006 Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum Catherine Lavazec, Sohini Sanyal and Thomas J. Templeton* Department of Microbiology and Immunology, Weill Cornell Medical College and the Program in Immunology and Microbial Pathogenesis, Weill Graduate School of Medical Sciences of Cornell University, New York, NY 10021, USA Received June 1, 2006; Revised October 18, 2006; Accepted October 20, 2006 ABSTRACT The human malaria parasite, Plasmodium falci- parum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte- trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM super- families, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus sup- porting the hypothesis that host immune pressure drives antigenic diversity within the loop. An exam- ination of multiple P.falciparum isolates demon- strates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence divers- ity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervaria- ble loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes. INTRODUCTION The bloodstream stages of the human malaria parasite, Plasmodium falciparum, reside enveloped by a parasitophorous vacuole within mature erythrocytes. As the parasite develops within this niche, it extensively modiﬁes the erythrocyte surface and cytoplasm in order to display parasite-encoded receptors on the erythrocyte surface as well as to establish erythrocyte surface solute channels that are involved in nutrient uptake and elimination of metabolic waste products. The requisite ‘extra-parasitic, intra-erythrocytic’ trafﬁcking network is of parasite origin and invention, because the ery- throcyte itself is devoid of trafﬁcking machinery that can be co-opted for parasitic means. Little is known regarding the molecular mechanisms of Plasmodium extra-parasitic traf- ﬁcking, but it likely involves components that are either soluble within the erythrocyte cytoplasm or integral within the parasitophorous vacuolar membrane; the Maurer’s clefts that dot the erythrocyte cytoplasm; or possibly small vesicles budding from either of these membrane surfaces [(1–7), reviewed in (8,9)]. Our understanding of protein targeting to the erythrocyte cytoplasm was recently given a revelatory boost by the identiﬁcation of a trafﬁcking motif that appears to be widely, but not inclusively, present in parasite-encoded, signal peptide-containing proteins that are trafﬁcked to the erythrocyte cytoplasm or surface (10–12). The motif is termed Pexel [Plasmodium export element, (12)] or VTS [vacuolar transport signal, (10)] and has a simple core consensus sequence, ‘RxLxQ/E’. All Pexel/VTS-containing proteins identiﬁed to date possess a two exon gene structure in which the signal peptide, oftentimes recessed from the start methionine, is encoded on the short N-terminal exon, whereas the Pexel/VTS motif is encoded on the second exon near the splice junction site [reviewed in (13,14)]. Using the above descriptive features of Pexel/VTS- encoding genes heuristically it is possible to compile a cata- log of P.falciparum proteins that are predicted to be trafﬁcked to the erythrocyte. This catalog most notably includes all proteins encoded by the 2 transmembrane (2TM)-containing members of the stevor, riﬁn and Pfmc-2TM gene families; the knob-associated proteins KAHRP and MESA; the DnaJ domain-containing RESA family; the superfamily of a-helical helical PHIST domain proteins (9) that contains over 70 members, including the RESA-like proteins; as well as a *To whom correspondence should be addressed. Tel: +1 212 746 4467; Fax: +1 212 746 4028; Email:tjt2001@med.cornell.edu The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors 2006 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.