Nucleic Acids Research, 2006, Vol. 34, No. 22 6699 Figure 1. Schematic representation of the catalog of P.falciparum Pexel/VTS-containing proteins. Signal peptide sequences are represented by gray boxes; transmembrane domains are represented by black boxes. All proteins are encoded by two exon genes; the location of the intron is represented by a gray triangle. The Pexel/VTS motif is encoded near the 50 end of the second exon and is represented by a narrow open box. The locations of signal peptide cleavage sites have not been determined, and the N-termini of mature proteins are not known. Predicted globular domains are indicated by open boxes. (Supplementary Figure 2) and 73 Riﬁn (Supplementary Figure 3) proteins also support a 2TM topology and conserved architectures consisting of: (i) highly conserved amino termini regions within the respective families, as well as similarity between Riﬁns and Stevors near the amino terminus; (ii) a hypervariable region between two con- served TM domains and (iii) a highly conserved, positively charged, short C-terminal domain (29). In contrast to the Stevor proteins, the Riﬁn N-terminal-most TM domain is atypical and contains an abundance of glycine and alanine residues, as well as a central proline residue that is conserved in a predominant subset of Riﬁns and is suggestive of a rela- tionship with ion channels (15). Cellular localization of the 2TM superfamilies For a given 2TM family, the pronounced sequence variability within the region between the TM domains, when contrasted with the relative sequence conservation in the N-terminal and C-terminal regions, suggests that this region deﬁnes a loop that is exposed to antibody-mediated immune pressure at the external surface of the erythrocyte. As a logical conclu- sion, it has been proposed that the erythrocyte surface is the ﬁnal trafﬁcking destination for the 2TM proteins and that they conduct their principal function at this site (28,29). However, the cellular localization of the 2TM super- family remains speculative and only the Riﬁn proteins have been shown to be exposed at the erythrocyte surface (30), whereas the Stevor and Pfmc-2TM proteins have been described as Maurer’s clefts proteins (28,31,32). To deter- mine the cellular localization of these proteins we generated transgenic NF54 parasites that abundantly express either Stevor (SFM, Stevor-FLAG-myc transgenic line) or Pfmc- 2TM (2TMFM, Pfmc2TM-FLAG-myc transgenic line) pro- teins, each of which were tagged with three c-myc epitopes at the carboxyl terminus plus two FLAG epitopes within the hypervariable loop (Figure 2A). IFA microscopy studies using anti-c-myc and anti-FLAG antibodies showed that the tagged proteins were efﬁciently expressed within the cyto- plasm of infected erythrocytes (Figure 2B); and both antibod- ies gave staining patterns that likely correspond to Maurer’s cleft localization, as described (28,31). The IFA studies of the chimeric transgenic proteins did not have sufﬁcient resolution to determine if these proteins are transported beyond the Maurer’s clefts to the erythrocyte surface; therefore, we used immunoelectron microscopy with anti-c-myc antibodies, which gave a stronger and more spe- ciﬁc staining by IFA than anti-FLAG antibodies, to examine the cellular localization of the epitope-tagged proteins. In both the SFM and 2TMFM transgenic lines numerous gold particles were associated with membranous structures that likely correspond to Maurer’s clefts, thus conﬁrming the above immunoﬂuorescence microscopy, as well as published data [(Figure 3a,g,h); (28,31,32)]. In addition, gold particles were associated with the erythrocyte membrane, and in a few instances associated with knobs, supporting the hypothe- sis that the 2TM proteins pass through Maurer’s clefts en route to their ﬁnal destination, the erythrocyte surface. How- ever, a small amount of protein (2–3 gold particles or clusters per thin section of erythrocyte) was observed in association with the surface; and we were not able to detect the tagged proteins by IFA on live cells using anti-FLAG antibodies (data not shown), although these proteins were abundantly expressed in both the SFM and 2TMFM transgenic lines. The relative abundance of gold particles, as well as of IFA ﬂuorescence, in Maurer’s clefts versus the erythrocyte mem- brane suggests that Maurer’s clefts might function to regulate the level of surface expression, in addition to a role in traf- ﬁcking. Moreover, it might explain the lack of surface expres- sion in studies that describe the Stevor and Pfmc-2TM proteins as Maurer’s cleft residents (28,31,32). It is currently being addressed if the inefﬁciency of surface transport is partially a phenotype of the NF54 isolate, and if greater erythrocyte surface display can be achieved using other trans- genic P.falciparum isolates, such as IT4. Nonetheless, we show that the 2TM proteins are trafﬁcked to the erythrocyte membrane and that the hypervariable region between two conserved TM domains likely deﬁnes a loop that is exposed on the erythrocyte surface. Diversity within the hypervariable loop of the 2TM superfamily members To implicate if immune pressure selects for divergence within the loop region and shapes an antigenically-diverse gene repertoire, we determined the sequence diversity between paralogs and across isolates for the Pfmc-2TM and Stevor families. Genomic DNA PCR products were isolated and