A complex of three related membrane proteins is conserved on malarial merozoites

Abstract

Invasion of human red blood cells by the malaria parasite Plasmodium falciparum is a coordinated, multi-step process. Here, we describe three novel integral membrane proteins that colocalize on the inner membrane complex immediately beneath the merozoite plasma membrane. Each has six predicted transmembrane domains and is conserved in diverse apicomplexan parasites. Immunoprecipitation studies using specific antibodies reveal that these proteins assemble into a heteromeric complex. Each protein was also expressed on insect cells using the baculovirus vector system with a truncated SUMO tag that facilitates maximal expression and protein purification while permitting cleavage with SUMO protease to release unmodified parasite protein. The expressed proteins were successfully reconstituted into artificial liposomes, but were not recognized by human immune sera. Because all three genes are highly conserved in apicomplexan parasites, the complex formed by their encoded proteins likely serves an essential role for invasive merozoites.

Fig. 1. Identification of the M6T family

(A) Membrane topology of PfM6Tα with each residue represented by a bead. The positions of the 6 predicted transmembrane domains, labeled with roman numerals, were determined using the TMHMM 2.0 algorithm. Residues conserved between the three P. falciparum paralogs are indicated using blue or red beads to represent identity between 2 or all 3 paralogs, respectively. (B) Unrooted Bayesian phylogeny of the apicomplexan M6T sequences. Posterior probabilities are given above or below the branches or using a curly bracket. The scale bar for branch lengths in this tree represents the number of expected changes per site [15]. Note the clear separation of the three paralogous clades.

Fig. 2. PfM6T proteins localize to the merozoite IMC

Confocal immunofluorescence images demonstrating location of each paralog, as indicated above each image. The top two rows of images each show a separate schizont-infected erythrocyte containing many merozoites; overlay of the red and green channels (rightmost column) shows colocalization of PfM6Tα with each other paralog. The bottom row shows each protein’s localization on spontaneously released merozoites. Red and green images reflect detection using specific antibodies raised in rabbits and mice, respectively. White scale bars represent 1 µM.

Fig. 3. PfM6T does not colocalize with MSP-1 or AMA-1

(A) IFA images of schizont-infected erythrocytes or freed merozoites (top and bottom rows, respectively) showing labeling with antibodies against PfM6Tα (red) and MSP-1 (green). Poor colocalization of these proteins is shown in the overlay of these images. (B) Quantification of label intensities along the parasite apical-posterior axis using single cells probed with two antigen-specific antibodies as indicated in each panel. Each x-y plot shows fluorescence intensity in arbitrary units at each point along the white line superimposed on the confocal image as a function of distance from point A. These positional intensities suggest that MSP-1 is external to each of the PfM6T proteins, which colocalize with each other precisely. (C) Freed merozoites demonstrating AMA-1 is restricted to the apical end of merozoites (green), but that PfM6Tα has a diffuse surface distribution (red). There is reduced PfM6Tα labeling at the apical end of each cell (overlay). Scale bars in A and C represent 1 µm. Similar results were obtained for PfM6Tβ and PfM6Tγ (not shown).

Fig. 4. PfM6T proteins localize on the IMC

Immuno-electron micrographs of merozoites probed with antibodies against PfM6Tα or MSP-1 (A and B, respectively) and detected with secondary antibodies conjugated to nanogold particles. In each row, the first two images were obtained with pre-embed antibody labeling of intact merozoites, while the rightmost image used antibodies applied after embedding and sectioning to permit access to intracellular antigens. Note that MSP-1 is recognized on intact merozoites, whereas PfM6Tα is not. Sectioning shows MSP-1 is on the parasite plasma membrane while PfM6Tα is on the IMC. Black scale bars in each image represent 100 nm. IMC, inner membrane complex; P, parasite plasma membrane.

Fig. 5. PfM6T proteins are simultaneously expressed and form a heterocomplex

Immunoblots showing specific detection of each PfM6T paralog as indicated above each group. In each group, lanes 1–3 were prepared from infected erythrocytes as a total lysate (lane 1), membrane fraction (lane 2), and soluble fraction (lane 3). Lane 4 corresponds to a total lysate prepared from uninfected erythrocytes. (B) Stage specific expression of each paralog, determined using immunoblots of parasite membranes harvested at indicated timepoints in h after synchronization. Each protein is maximally expressed on schizonts and merozoites. (C) Immunoblots demonstrating co-precipitation of each paralog with PfM6Tα and PfM6Tβ (first and second groups) and not with RAP1 (third group). The first lane in each group is a control that shows blotting of total infected cell lysate without precipitation. Subsequent lanes reflect protein eluted from beads conjugated to antibodies against indicated paralog or without antibodies (“—”). Positions of molecular weight standards (in kDa) are indicated to the right of each blot.

Fig. 6. Expression on insect cells

(A) Schematic representation of the engineered chimeric protein. The N-terminal tag (6-His followed by a 53 residue CTHS) increases the expected molecular weight of the fusion protein by approximately 6 kDa. This tag can be removed by SUMO protease digestion (arrow). (B) Immunoblots showing detected expression of each 6-His-CTHS-PfM6T chimera. In each group, total lysate from untransfected Sf9 cells (“Un”) or from transfected cells that express a single PfM6T paralog as indicated in the top row above each blot was loaded and probed with either paralog-specific antibody or anti-SUMO antibody recognizing the CTHS tag (“S”, bottom row above blot). Each specific antibody recognizes only cells expressing the corresponding protein. (C) Immunoblots showing reconstitution of expressed proteins, enriched by Ni²⁺-NTA purification from T. ni extracts, into azolectin liposomes (lane 2). In each blot, an ultracentrifugation supernatant (lane 1) revealed negligible amounts of soluble protein. (D) Liposome-reconstituted PfM6Tβ before and after cleavage of the 6-HIS-CTHS tag by SUMO protease to yield the native PfM6Tβ (lanes 1 and 2, respectively). The molecular weight of the cleaved protein is 42 kDa, matching that of the in situ parasite protein. The positions of molecular weight standards (in kDa) are indicated to the right of each blot.