Mutant/mutation
The mutant expresses a C-terminal 3xcmyc-tagged version of SMAC and expresses GFP-luciferase under the constitutive eef1a promoter

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.05.28.596273

Protein (function)
In P. berghei evidence has been presented that SMAC is a protein exported into the cytoplasm of infected erythrocytes and plays a role in CD36-mediated sequestration of schizonts in capillaries of inner organs. P. berghei SMAC  contains a signal sequence, a PEXEL motif (RFLVE) and a transmembrane domain. Orthologs are only found in the murine Plasmodium species. Staining of  live SMAC::mCherry and wild type infected erythrocytes with anti-mCherry or anti-SMAC antibodies did not provide evidence for exposure of SMAC on the surface of infected erythrocytes.

Phenotype
SMAC-myc was concentrated at the iRBC periphery. It is present in distinct foci seemingly lining the inside of the iRBC.

Additional information
To determine the subcellular location of EMAP3 we generated a P. berghei mutant with a C-terminal triple-myc tag in the endogenous emap3 locus (RMgm-5574). In addition, we generated P. berghei mutants with triple-myc tagged EMAP2 (PBANKA_0836800; RMgm-5575) or EMAP1 (PBANKA_0316800; RMgm-5576) proteins that are known to be exported to the iRBC membrane or SMAC (PBANKA_0100600; RMgm-5577), which is known to be involved in P. berghei iRBC sequestration.

In schizonts EMAP3-myc and EMAP1-myc show a location largely concentrated to the iRBC membrane, while EMAP2-myc displayed a more diffuse localisation throughout the iRBC cytoplasm. SMAC-myc was also concentrated at the iRBC periphery but, in contrast to EMAP3 and EMAP1, it is present in distinct foci seemingly lining the inside of the iRBC.
To further investigate the location and timing of expression of EMAP3 we took samples from cultures containing roughly synchronised blood stages (ring, early trophozoite, late trophozoite, and schizont) and performed IFA to detect EMAP3-myc and EMAP1-myc. EMAP3 is expressed already in ring forms, where it appears closely associated with the nucleus (likely still residing in or close to the endoplasmic reticulum, ER).  Already in early trophozoites EMAP3-myc is found at the iRBC membrane and EMAP3 remains located at the iRBC membrane in schizonts. The active export of EMAP3 and the specificity of the EMAP3-myc signal at the iRBC membrane was verified by treatment with brefeldin A (BFA), which traps parasite proteins during transport between the ER and Golgi apparatus. As expected, BFA treatment inhibited export of EMAP3-myc and abolished EMAP3-myc iRBC membrane staining in mature parasite stages (schizonts/late trophozites). In younger parasite stages (rings), EMAP3-myc accumulated in close proximity to the parasite ER (visualised by co-staining of the ER-resident protein BiP (binding immunoglobulin protein), in both control and BFA treated parasites. EMAP3-myc expression and location closely mirrors that of EMAP1-myc, which is also exported to the iRBC membrane in trophozoites and schizonts.  A notable difference is that EMAP1-myc is not detectable in ring forms.

How EMAP3 is is oriented on the iRBC membrane was identified through surface shaving using trypsin and permeabilisation with Triton X-100. EMAP3-myc is detectable without prior permeabilisation, demonstrating that the C-terminal end of the protein containing the myc tag is exposed on the iRBC surface. This is supported by the observation that the EMAP3-myc signal is lost upon treatment with trypsin. In contrast, EMAP1-myc could only be detected when the cell is permeabilised, indicating that the C-terminus of EMAP1 is not exposed on the surface of the iRBC.  Despite its association with the iRBC membrane, EMAP1 lacks predicted transmembrane domains and the orientation of the protein at the iRBC membrane was not further studied.

To analyse the orientation and location at the iRBC membrane we stained the iRBC with BODIPY TR ceramide to visualise membranes and/or N-hydroxysuccinimide (NHS)-ester that binds to protein dense regions.  UEx-M analysis confirms that EMAP3-myc localises to the iRBC membrane in mature schizonts, where it co-localised with the BODIPY-stained RBC membrane. EMAP1-myc could also be observed in close proximity to the iRBC membrane, however the EMAP1-myc staining in UEx-M was more widespread and was present more widely across the host cell cytoplasm. In addition, in agreement with IFA results, SMAC-myc was concentrated in distinct patches, seemingly located just under the iRBC membrane. Taken together, these analyses show that EMAP3 is exported into the iRBC where it is anchored as a multi-pass transmembrane domain protein with its C-terminus exposed on the surface of the iRBC.

Evidence is presented for:
- EMAP3 interacts with EMAP1 but not with SMAC at the iRBC membrane

Other mutants