A novel family of Apicomplexan glideosome-associated proteins with an inner membrane-anchoring role

Abstract

The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.

FIGURE 1.

GAPMs form an Apicomplexan-specific protein family characterized by six transmembrane domains and short cytoplasmic loops/tails. A, a multiple alignment of the protein sequences of the P. falciparum and T. gondii GAPM sequences with their amino acids shaded according to their side chain characteristics. Transmembrane domains were identified using TMHMM (version 2.0) (29). TM, transmembrane domain; L, loop. B, neighbor-joining tree constructed using all available amino acids sequences of GAPM family members. Evolutionary distances are estimated under the JTT model. Calculated bootstrap values are indicated on significant branches. Each GAPM protein falls into one of three distinct orthologous groups; GAPM1, -2 or -3. Diagrams indicating predicted GAPM membrane orientation predicted by TMHMM are shown beside each GAPM group.

FIGURE 2.

GAPM proteins localize to the IMC. A, immunofluorescence images of PfGAPM1-HA and PfGAPM3-HA expressing P. falciparum schizonts probed with an anti-HA antibody and antibodies to either the IMC-resident protein PfGAP45 or the MSP1₁₉ subunit of the plasma membrane protein MSP1. PfGAPM1-HA and PfGAPM3-HA co-localize with PfGAP45 but not with MSP1₁₉. B, TgGAPM-ChFP-HA-expressing parasites were treated with C. septicum α-toxin to swell the parasite membrane away from the IMC. Probing fixed parasites with anti-HA and the parasite membrane marker SAG1 validates that TgGAPM proteins reside in the IMC. Scale bar, 1 μm.

FIGURE 3.

P. falciparum GAPM proteins are present in oligomeric complexes resistant to disassociation. A, Western blots of schizont stage transgenic parasites solubilized with detergent mixes of different strengths (1% TX-100 < RIPA < 2% SDS < 2D) indicate that PfGAPM1-HA is the most resistant to solubilisation, PfGAPM3-HA is intermediate and PfGAPM2-GFP the most easily solubilized. Other known IMC proteins, PfGAP45 and PfGAP50, are easily solubilized by all detergent treatments. Cytoskeletal ALVs are most readily solubilized by the harsher detergent treatments. B, cross-linking parasite proteins with DSP indicated that the PfGAPMs form insoluble high molecular weight oligomers. Mono- and dimeric forms of the PfGAPMs are more soluble, and the majority of the GAPM protein remained in the insoluble pellet fraction. NR, non-reduced; R, reduced.

FIGURE 4.

GAPM proteins interact with components of the actin-myosin motor and subpellicular network. PfGAPM1-HA (A) and PfGAPM3-HA (B) were immunoprecipitated with anti-HA beads from parasites expressing the tagged proteins. To ensure the precipitations worked, Western blots were probed with anti-HA. Probing the precipitates with rabbit anti-GAP45, anti-GAP50, and anti-ALV repeat indicated that these proteins were binding to PfGAPM1-HA and to a lesser extent to PfGAPM3-HA. D, PfGAPM2-GFP-expressing parasites were immunoprecipitated with anti-GFP beads and Western blots were probed as previously described. GAP45 and -50 and particularly ALV4 appeared to bind to PfGAPM2-GFP. C and E, the validity of these interactions was supported by failure to precipitate any of these proteins from wild type 3D7 parasites with anti-HA and anti-GFP beads. F–H, anti-HA immunoprecipitations were performed on tachyzoites expressing TgGAPM-1/2/3-HA-ChFP and Western blots of these probed with anti-HA certified the precipitations worked. The Western blots were also probed with anti-GAP45 and anti-ALV repeat, which indicated these proteins co-immunoprecipitated with the TgGAPMs. I, control immunoprecipitations with wild-type RH parasites and anti-HA beads were unsuccessful for GAP45 but small amounts of a protein that bound the ALV-repeat antibody was precipitated. The arrowhead indicates a non-specifically binding ALV band.

FIGURE 5.

Reciprocal immunoprecipitation assays indicate that the alveolins and GAP45 interact with GAPM proteins. Western blots of alveolin and GAP45 immunoprecipitation assays on lysates from P. falciparum schizonts expressing either PfGAPM1-HA (A), PfGAPM2-GFP (B), or PfGAPM3-HA (C) were probed with antibodies listed in individual panels. Probing with anti-ALV or anti-GAP45 confirmed that immunoprecipitations were successful. Probing with either anti-HA (A and C) or anti-GFP (B) indicated that each GAPM co-purified with the alveolins and GAP45. The arrow represents the anti-ALV IgG band.

FIGURE 6.

Proposed model of GAPM interaction. GAPM proteins reside within the IMC and have been shown to interact with components of the actin-myosin motor and subpellicular network. The plus signs represent the solubility of each of the proteins in each buffer as judged by Western blotting: +, proteins readily soluble in listed buffer; ++++, proteins poorly soluble in listed buffer.