Summary

RMgm-680
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0524200; Gene model (P.falciparum): Not available; Gene product: small exported protein early transcribed membrane protein (SEP2; ETRAMP; Pbsep2; ETRAMP4)
Name tag: FLAG
Phenotype Asexual bloodstage;
Last modified: 26 December 2011, 22:19
  *RMgm-680
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22106924
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 8417HP
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (Janse et al., Exp. Parasitol. 68, 274-282).
The mutant parasite was generated by
Name PI/ResearcherCurà, C; Pace, T; Ponzi, M.
Name Group/DepartmentDipartimento di Malattie Infettive, Parassitarie ed Immunomediate
Name InstituteIstituto Superiore di Sanità
CityRome
CountryItaly
Name of the mutant parasite
RMgm numberRMgm-680
Principal nameF-sep2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageFLAG-tagged SEP2 (SEP2-F) expressed in blood stages. SEP2-F was located at the periphery of parasites, and in punctuate structures within the host cell cytoplasm.
See 'Additional remarks phenotype'.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a (C-terminal) FLAG-tagged version of SEP2. The endogenous sep2 gene is not tagged. The FLAG-tagged copy of sep2 is introduced into the genome into the (silent) c/d-ssu-rRNA locus. The FLAG-tagged copy of sep2 is under the control of the upstream and downstream regulatory regions of the endogenous sep2.

Protein (function)
Early transcribed membrane protein (ETRAMP) family member. Plasmodium conserved family with greater than ten members in P. falciparum. ETRAMPs are abundantly expressed early in the intraerythrocytic cycle and are small (frequently less than 200 aa) integral membrane proteins that are localized within the parasitophorous vacuolar membrane (PVM). All members have signal peptides plus a transmembrane domain. The ETRAMP/SEP proteins of P. yoelii and P. berghei (8-11 genes) show homology to members of the ETRAMP family of proteins of P. falciparum (14 genes) but the orthologous relationship of the different members is not completely resolved.

P. berghei sep genes, Pbsep1, Pbsep2 and Pbsep3 (PBANKA_052480, PBANKA_052420 and PBANKA_050110, respectively) are 3 ETRAMP members which reside in the subtelomeric regions of chromosome 5. The three genes share the upstream regulatory region and differ in their 3’UTRs. The encoded proteins (13-16 KDa) are nearly identical in the first 81 amino acids, which include a predicted signal peptide, a lysine-rich domain and a TM region, while differ in their C-terminal portion.
Mutants lacking expression of SEP1 have been generated (RMgm-16, RMgm-18) which show a normal phenotype during the complete life cycle, comparable to wild type parasites.
Multiple unsuccessful attempts to disrupt sep2 en sep3 (RMgm-64, RMgm-65) indicate that these proteins are essential for blood stage proliferation.
SEP1 is an integral membrane of the parasitophorous vacuole membrane (PVM) of blood stages.

Phenotype
Antibodies against SEP2 recognized the endogenous SEP2 and the FLAG-tagged version in Western analysis. SEP2-F was located at the PVM, and in punctuate vesicle-like structures within the host cell cytoplasm. Immuno-electron microscopy provided evidence that the C-terminus of SEP2 in the PVM face the RBC cytoplasm.
See RMgm-681 for analyses of mutants expressing FLAG-tagged SEP3, showing transport  of SEP3 into the cytoplasm of the host erythrocyte.
For both SEP2 and SEP3 it is shown that these proteins are components of the parasitophorous vacuole membrane (PVM). During blood stage development vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton.

Additional information
In the paper additional analyses are shown for determination of motifs for export of these proteins using transgenic parasites that express GFP under the control of motifs/sequences of sep2 and sep3.
'To define protein motifs which mediate the export of SEP proteins,  different transfection constructs were designed, which included the fluorescent GFP reporter. Transgenic parasites, generated after transfection, were analyzed by fluorescence microscopy to assess subcellular localization of the GFP. The common 1,2 kb-region upstream the coding region of sep genes and the specific 3’UTRs expressed the reporter GFP in the cytoplasm of all parasite blood stages indicating that the variable 3’UTRs do not modulate expression/stability of sep transcripts. We then expressed the GFP appended to growing portions of SEP2 coding region under the control of Pbsep2 specific 3’UTR. The predicted N-terminal SP, contained in the first 28 amino acids, routed the GFP to the parasite periphery. The reporter was partially retained in a perinuclear localization, indicative of the endoplasmic reticulum (ER). A similar localization of the reporter was observed when the next highly charged lysine-rich region (25 amino acids) was added to the construct. The ER localization of the chimeric proteins was confirmed by IFA experiments using antibodies specific for the ER marker BIP and a anti-GFP immune serum. At difference, when the SP was removed, the internal hydrophobic region was not able to route the chimeric protein to the parasite periphery. The GFP fluorescence was, in fact, detected exclusively inside the parasite. This suggests that the internal TM does not act as a recessed signal peptide. When the GFP was appended to the first 81 aminoacids, which include both SP and TM, the chimera was efficiently exported to the erythrocyte cytoplasm. The GFP signal at the PVM was, however, less intense than that observed in IFA using SEP2-specific immune serum. A possible explanation is that the presence of the GFP moiety may interfere with the correct localization of the chimeric protein to this membrane compartment. Overall these findings suggest that SEP2 export is a two-step process, the protein is first routed to the PVM via ER and then translocated to the host cell compartment'.

Other mutants
See RMgm-681 for more detailed analyses of a mutant expressing FLAG-tagged SEP3


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0524200
Gene Model P. falciparum ortholog Not available
Gene productsmall exported protein early transcribed membrane protein
Gene product: Alternative nameSEP2; ETRAMP; Pbsep2; ETRAMP4
Details of the genetic modification
Name of the tagFLAG
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationSee the paper for details of the constructs, (sequence of) primers and methods used to tag sep1 with FLAG
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6