A Plasmodium gene family encoding Maurer's cleft membrane proteins: structural properties and expression profiling

Abstract

Upon invasion of the erythrocyte cell, the malaria parasite remodels its environment; in particular, it establishes a complex membrane network, which connects the parasitophorous vacuole to the host plasma membrane and is involved in protein transport and trafficking. We have identified a novel subtelomeric gene family in Plasmodium falciparum that encodes 11 transmembrane proteins localized to the Maurer's clefts. Using coimmunoprecipitation and shotgun proteomics, we were able to enrich specifically for these proteins and detect distinct peptides, allowing us to conclude that four to 10 products were present at a given time. Nearly all of the Pfmc-2tm genes are transcribed during the trophozoite stage; this narrow time frame of transcription overlaps with the specific stevor and rif genes that are differentially expressed during the erythrocyte cycle. The description of the structural properties of the proteins led us to manually reannotate published sequences, and to detect potentially homologous gene families in both P. falciparum and Plasmodium yoelii yoelii, where no orthologs were predicted uniquely based on sequence similarity. These basic proteins with two transmembrane domains belong to a larger superfamily, which includes STEVORs and RIFINs.

Figure 1

Colocalization immunofluorescence of PfMC-2TM with Pf130. Trophozoite and schizont-infected erythrocytes were fixed with ice-cold acetone and incubated sequentially with the primary antibodies SP1C1 and SP1A6 specific for PfMC-2TM and Pf130, respectively. The monoclonal antibodies were contained in spent hybridoma culture supernatant and were used undiluted. The anti-mouse secondary antibodies conjugated to Alexa 488 and Alexa 568 (Molecular Probes) were diluted 1: 1000 in 1× PBS and added separately. Following incubation for 1 h at 37°C, slides were washed three times in 1× PBS and once in distilled water. The smears were mounted in Vectashield (Vector) containing 4′, 6-diamidino-2-phenylindole (DAPI) to stain parasite DNA. Parasites were examined by a Nikon epifluorescence microscope. (A) P. falciparum-infected erythrocytes incubated with mAb SP1A6 followed by anti-mouse 488 Alexa conjugate (green). (B) P. falciparum schizont-infected erythrocytes incubated with mAb SP1C1, followed by anti-mouse 568 Alexa conjugate (red). (C) Parasite nuclei counterstained by DAPI. (D) Overlay of SP1A6 and SP1C1 antibodies showing colocalization (yellow) of PfMC-2TM and Pf130 Maurer's cleft proteins.

Figure 2

Multiple sequence alignments. The sequences of the Maurer's cleft 2-transmembrane domain proteins were aligned using CLUSTAL W (1.82) (Thompson et al. 1994). (*) Conserved residues. Bold, underlined sequences indicate regions of the proteins that were extended from the originally published gene models (Gardner et al. 2002), on the basis of the complete chromosome sequences that were obtained from PlasmoDB (Bahl et al. 2002). The red-dotted vertical line indicates the boundary between exon 1 and exon 2. The regions covered by peptides identified during the MS/MS analyses are highlighted. The green, magenta, cyan, and yellow color coding stands for peptides unique to a protein, and peptides common to two, three, and more than three proteins, respectively. The green boxes indicate the position of signal peptide and transmembrane domains as predicted by SignalP (Nielsen et al. 1997) and TMHMM (Krogh et al. 2001), respectively. The arrow points at the potential processing site. The red and blue boxes define potential bipartite vacuolar translocation signals (VTSs) as described by Lopez-Estrano et al. (2003).

Figure 3

Schematic representation of the Plasmodium falciparum 2-transmembrane proteins encoded by subtelomeric multicopy gene families. The number of genes and known subcellular localizations (ES, erythrocyte surface and MC, Maurer's Cleft), as well as the average number of amino acid residues in the extracellular loop are reported for the RIFIN, STEVOR, and PfMC-2TM families.