| 2011 |
CLAG3.1 (PF3D7_0302500) and its paralog clag3.2 are the parasite genes responsible for PSAC (plasmodial surface anion channel) activity at the host erythrocyte membrane; DNA transfection and in vitro selection experiments demonstrated that PSAC-inhibitor interactions are encoded by the clag3 gene family, and the encoded protein inserts as an integral protein in the host membrane, increasing host cell permeability to diverse solutes. |
High-throughput nutrient-uptake inhibitor screening, genetic cross linkage analysis (Dd2 × HB3), DNA transfection, in vitro selections, inhibitor specificity assays |
Cell |
High |
21620134
|
| 2001 |
CLAG3.1 (PF3D7_0302500; encoded by PFC0120w on chromosome 3) is identified as a component of the high-molecular-mass RhopH1 subunit of the merozoite rhoptry RhopH complex; peptide sequencing by mass spectrometry and Edman degradation of monoclonal antibody-purified RhopH complex identified chromosome-3 clag family members as encoding RhopH1 in P. falciparum. |
Monoclonal antibody purification of RhopH complex, peptide mass spectrometry, Edman degradation sequencing |
Molecular and biochemical parasitology |
Medium |
11738712
|
| 2005 |
CLAG3.1 protein localizes to the apical end of merozoites in segmented schizonts and is a component of the PfRhopH complex; immunoprecipitation with anti-Clag9 antibody did not co-precipitate CLAG3.1, indicating that individual PfRhopH complexes contain only one RhopH1/clag gene product at a time. |
Specific antisera localization (immunofluorescence), immunoprecipitation of PfRhopH complex with anti-Clag9, anti-Clag2, and anti-Clag3.1 antibodies |
Molecular and biochemical parasitology |
Medium |
15953647
|
| 2013 |
CLAG3.1 and CLAG3.2 undergo mutually exclusive, epigenetically regulated expression switching; selection with low-dose blasticidin S caused parasites to switch from clag3.2 to clag3.1 expression, demonstrating that the two clag3 isoforms confer different solute transport efficiencies to PSAC. Simultaneous silencing of both clag3 genes severely reduced PSAC-mediated uptake. Switches were not accompanied by genetic mutations at the clag3 loci. |
Drug selection (blasticidin S at varying concentrations), solute uptake assays, RT-PCR expression analysis, whole-genome analysis for mutations |
Cellular microbiology |
Medium |
23819786
|
| 2011 |
Silencing of clag3 paralogs (including clag3.1) is associated with enrichment of heterochromatin-associated histone modifications, as shown by chromatin immunoprecipitation. Parasites with both clag3.1 and clag3.2 silenced (clag3-null) exhibit significant growth inhibition, providing genetic evidence that clag3 gene products are required for efficient parasite proliferation. |
Chromatin immunoprecipitation (ChIP) for histone modifications, gene disruption of clag3.2 locus, drug-selection-mediated silencing of clag3.1, growth assays |
Molecular microbiology |
Medium |
21320181
|
| 2018 |
CLAG3.1 (PF3D7_0302500) undergoes self-association (oligomerization) at the host erythrocyte membrane. Engineering parasites to express two CLAG3 isoforms simultaneously showed stoichiometric trafficking of both to the host membrane and yielded intermediate PSAC channel phenotypes. Co-immunoprecipitation and surface labeling confirmed CLAG3 oligomer formation. In vitro selections produced mutants with correlated changes in CLAG3 sequence and channel activity, supporting CLAG3 oligomers as the channel-forming unit. |
Transfection to co-express two CLAG3 isoforms, co-immunoprecipitation, surface biotin labeling, electrophysiological patch-clamp (PSAC phenotype), in vitro inhibitor selection |
mBio |
High |
29739907
|
| 2020 |
CLAG3 conditional knockdown in a genetic cross where one parent carries a single clag3 gene confirmed CLAG3's primary role in PSAC nutrient transport. However, complete CLAG3 knockout did not abolish transport activity under standard culture conditions (unlike RhopH2/RhopH3 knockouts), but the CLAG3 knockout parasite failed to propagate in medium resembling human plasma, indicating CLAG3 is required under physiological nutrient conditions. These results implicate oligomerization of CLAG paralogs from other chromosomes in channel formation. |
Conditional knockdown (TetR-DOZI aptamer system), complete knockout by genetic disruption, linkage analysis in P. falciparum genetic cross, nutrient/solute uptake assays, propagation in modified (physiological) medium |
PLoS pathogens |
High |
32069335
|
| 2020 |
Live-cell FRET imaging showed that CLAG3 and RhopH2 remain physically associated from rhoptry organelles through merozoite egress, parasitophorous vacuole deposition, and export to the infected erythrocyte membrane; the two proteins traffic together without dissociation throughout this entire journey. Whole-cell patch-clamp demonstrated that doubly-infected erythrocytes have twice the PSAC functional copy number, establishing a quantitative (stoichiometric) relationship between RhopH complex copy number at the membrane and channel activity. |
Live-cell FRET (mCerulean/mVenus-tagged CLAG3 and RhopH2), fluorescence imaging of intracellular trafficking, whole-cell patch-clamp electrophysiology |
mBio |
High |
32900800
|
| 2022 |
During merozoite invasion, CLAG3.1 (as the RhopH complex component Clag3) is released directly into the host cell cytoplasm, while RhopH2 and RhopH3 are released into the nascent parasitophorous vacuole. Assembly of the Clag3/RhopH2/RhopH3 complex occurs only after export of RhopH2 and RhopH3 from the parasitophorous vacuole into the erythrocyte cytoplasm, followed by incorporation into the host cell membrane concomitant with nutrient uptake activation. This compartmentalization prevents premature channel assembly. |
Super-resolution microscopy, conditional knockdown, immunofluorescence localization, co-immunoprecipitation during defined invasion stages |
Communications biology |
High |
35393572
|
| 2016 |
Biochemical fractionation and native PAGE of CLAG3.1-expressing infected erythrocyte membranes identified at least three distinct high-molecular-weight complexes containing CLAG3: ~720 kDa (CLAG3 + RhopH2 + RhopH3), ~620 kDa (CLAG3 + RhopH2), and ~480 kDa (CLAG3 alone, likely a homo-oligomer). Extracellular biotin labeling and protease susceptibility studies localized the ~480 kDa CLAG3-only complex to the erythrocyte membrane surface, suggesting it may represent the functional pore unit of PSAC. |
Co-immunoprecipitation from membrane fractions, native PAGE, mass spectrometry, extracellular biotin labeling, protease protection/susceptibility assays |
PloS one |
Medium |
27299521
|
| 2006 |
The N-terminal one-third of CLAG3.1, which contains a conserved domain shared with Toxoplasma gondii RON2, cannot associate with the RhopH complex when expressed as a GFP chimera; however, a c-Myc-CLAG3.1 chimera lacking the C-terminus successfully associates with the RhopH complex, indicating that the middle region of CLAG3.1 is required for RhopH complex association but the C-terminus is not necessary. |
Transgene expression constructs in P. falciparum, co-immunoprecipitation of chimeric proteins with RhopH complex |
Parasitology international |
Medium |
17175193
|
| 2005 |
Synthetic peptides spanning the CLAG3.1 sequence were tested in cell-binding assays; twelve peptides (HABPs) bound C32 cells (which express CD36) with high affinity (Kd 100–800 nM), and five of these also bound erythrocytes. Cross-linking and SDS-PAGE identified erythrocyte-binding proteins of ~26 kDa and ~59 kDa and a ~53 kDa protein as a possible receptor on C32 cells for CLAG3.1 peptides. |
Synthetic peptide binding assays (C32 cells and erythrocytes), affinity constant determination, cross-linking, SDS-PAGE |
Protein science |
Low |
15659379
|