Mutant/mutation
The mutant expresses a C-terminal 4xMyc-tagged version of MDR1 and a a N-terminal 6xHA-tagged version of UBP1. HA-tagging of UBP1 was performed in the mutant expressing Myc-tagged MDR1 (RMgm-5546).

Protein (function)
MDR1: Drug resistance in Plasmodium has been associated with single nucleotide polymorphisms and an increased copy number of the multidrug resistance protein 1 (MDR1), an ATP-binding cassette (ABC) protein family member. It is a P-glycoprotein that localizes to the digestive vacuole membrane and import solutes, including some antimalarial drugs, into the digestive vacuole.

UBP1: Mutations of V2697F and V2728F in a de-ubiquitinase (or ubiquitin carboxyl-terminal hydrolase 1, UBP1; PF3D7_0104300) have been linked to Plasmodium chabaudi responses to artesunate (ATS), artemisinin (ART), and/or chloroquine (CQ). Introductions of equivalent mutation of PcUBP1 V2697F into P. falciparum and P. berghei increased ART resistant levels and the introduction of the PcUBP1 V2728F equivalent mutation caused ART and CQ-resistance in P. berghei, but not in P. falciparum. PfUBP1 variants were associated with ART-delayed parasite clearance in P. falciparum isolates from Kenya and Thailand. A PfUBP1 R3138H substitution was shown to confer ART resistance in a ring-stage survival assay in vitro.

Phenotype
Both UBP1 and MDR1 were expressed in blood stages, gametocytes, gametes, zygotes, and ookinetes, but not in oocysts and sporozoites of the NSS(IP) parasite.
However, MDR1 colocalized with the digestive vacuole membrane marker CRT in the NSS(IP) parasite, but not with UBP1. Within the trophozoites having UBP1 NT haplotype, MDR1 was mostly detected on parasite plasmalemma membrane (PPM) as well as on the cytostome, an apparatus that mediated endocytic invagination for the uptake of host hemoglobin. Additionally, the MDR1 signals in the UBP1 NT parasites appeared to be surrounded by the parasitophorous vacuole membrane (PVM) protein SEP1. These results confirm the MDR1 localization on DVM in the NSSIP parasites and on PPM in the NSRNT and NSSNT parasites.

Additional information
Evidence is presented that:
- Mefloquine reistance is P. yoelii is associated with two mutated amino acids in UBP1: I1560N and P2874T located in the conserved central region (CCR) of UBP1. The CCR has some homology with the motifs III-IV of the glycosyltransferase (GTase) domain of bacterial penicillin-binding proteins (PBPs).
- UBP1 double mutations (NT) impair parasite growth, hemozoin formation, and de-ubiquitinase activity (RMgm-5544).
- UBP1 double mutations (NT) alters UBP1 localization pattern and aggregation. NT substitutions can affect UBP1 aggregation and distribution possibly by destabilizing hydrophobic interaction after the introduction of the two hydrophilic residues.
To study the UBP1 protein localization and expression in different parasite stages, we tagged the P. yoelii UBP1 with a sextuple hemagglutinin (6HA) at N-terminus and a variable coding region between GDVKT and VQQSG residues in both NSS and NSR parasites using CRISPR/Cas9 method (see RMgm-5545). UBP1 was expressed as punctate structures in the cytoplasm of asexual blood-stage trophozoites and schizonts; it was also expressed in gametocytes, gametes, zygotes, and ookinetes, but not in oocysts and sporozoites of the NSS-IP. UBP1 in the NSR(NT) or NSS(NT) mutant parasites was distributed more widely in the cytoplasm than NSS(IP) and NSS(IT) in the intra-erythrocytic stages. We engineered two additional parasite clones, NSS(IP/ubp1::GFP), and NSS (NT/ubp1::GFP), with ubp1 being endogenously tagged with a GFP coding sequence at the N-terminus. The GFP-tagged parasites further verified that NSS(NT) had a more diffused expression of UBP1 than NSS(IP) in the asexual blood stages. These data show that IP → NT substitutions can affect UBP1 aggregation and distribution possibly by destabilizing hydrophobic interaction after the introduction of the two hydrophilic residues.
- Plasmodium UBP1 NT substitutions increase MDR1 ubiquitination.
To investigate the impact of the altered MDR1 ubiquitination on protein expression and trafficking, we tagged the endogenous MDR1 in the NSS(IP), NSR(NT), and NSS(NT) parasites with quadruple Myc epitope (4Myc) at the C-terminus to obtain mdr1::4Myc parasites (RMgm-5546). Because PfCRT and PfK13 contribute to parasite responses to CQ, DHA, and other drugs, we also tagged P. yoelii K13 with 4Myc at the N-terminus and CRT with 3HA at the C-terminus obtaining the k13::4Myc and crt::3HA. Immunoblotting analysis confirmed that NT substitutions in the UBP1 rendered higher levels of MDR1 ubiquitination.
- UBP1 NT substitutions translocate MDR1 from the DV membrane to the plasma membrane.
In the NSS(IP) trophozoites,MDR1 was expressed in hemozoin-containing DV scattered in the cytoplasm; in schizonts, most of the MDR1 co-localized with large hemozoin. Similar pattern of MDR1 localization was observed in the NSS(NP) and NSS(IT) parasites. The MDR1 was mostly (more than 95% of cells) localized at the parasite plasma membrane (PPM) of trophozoites and schizonts in both NSR(NT) and NSS(NT) parasites. To confirm the altered MDR1 protein localization, we genetically engineered three doubly tagged parasites (DTPs), mdr1::4Myc/ubp1::6HA (RMgm-5547), mdr1::4Myc/crt::3HA and mdr1::4Myc/sep1::3HA from the mdr1::4Myc parasite (RMgm-5546). Parasite proteins CRT and SEP1 are natively localized to DVM and parasitophorous vacuole membrane (PVM) that surrounds the parasite PPM, respectively. These results confirm the MDR1 localization on DVM in the NSS(IP) parasites and on PPM in the NSRNT and NSSNT parasites.
- Variants of UBP1 display reduced Fluo-4 and MFQ accumulation in the parasite cytoplasm.
To investigate the substrate transport directions of the MDR1 in parasites carrying UBP1 IP and NT haplotypes, we engineered two parasite clones, NSS(IP/mdr1::mCherry)(RMgm-5548) and NSS(NT/mdr1::mCherry) that had the N-terminus of the MDR1 fused with mCherry protein.  As expected, mCherry fluorescence was detected specifically at DVM and PPM in NSS(IP) and NSS(NT), respectively. Next, we investigated the accumulation of a known PfMDR1 substrate Fluo-49,37 in the NSS(IP/mdr1::mCherry) and NSS(NT/mdr1::mCherry) parasites.

We  performed allelic exchanges of the two UBP1 mutations (I1560N and P2874T) between NSS and NSR lines to investigate the roles of the mutations in modulating drug response and parasite fitness. Two DNA fragments covering the mutation sites were amplified from the parasites: Fragment R1 was a 644 bp DNA having I1560N substitution, and fragment R2 was a 667 bp DNA harboring P2874T substitution. Two plasmids each containing the R1 or R2 sequence from NSS or NSR were cross transfected sequentially to replace IP with NT in the NSS parasite and NT with IP in the NSR parasite. Self-replacements (IP replaced with IP in NSS and NT replaced with NT in NSR) were also performed to serve as transfection controls, generating eight allelic replacements with four haplotype combinations, namely NS(SIP-self), NSS(NP), NSS(IT), NSS(NT), NSR(NT-self), NSR(IT), NSR(NP), and NS(RIP). Single replacement clonal parasites of NSS(NP), NSS(IT), NSR(IT), and NS(RNP), double replacement NSR(IP), self-replacement NSS(IP-self), and NSR(NT-self) were obtained from one to three rounds of transfection and pyrimethamine (Pyr) selection, but not NSS(NT) parasites. NSS(NT) clones were finally obtained after additional selection with 20mg/kg MFQ for 4 days, suggesting a disadvantage in vivo growth and MFQ-resistant trait of the NSS(NT) parasites. This result is consistent with the finding that NSR parasite grew slower than the NSS parasite without drug selection.
In vivo, drug assays testing parasite responses to MFQ, LUM, QN, PPQ, CQ, and DHA of the allelic exchanged parasites with NSS background showed that introduction of a single mutation, NSS(NP) or NSS(IT), had a slight increase in IC90 to MFQ, LUM, and PPQ, but not IC50 values or no effect on parasite responses to high levels of MFQ, LUM, and PPQ. In contrast, NSS(NT) parasites with double substitutions dramatically increased parasite survival after MFQ, LUM, and PPQ treatments. 
As expected, in the set of parasites with NSR background, both NSR parent and NSR(NT-self) were resistant to MFQ, LUM, and PPQ, while single replacements (NSR-IT and NSR-NP) and double replacement (NSR-IP) were sensitive to these antimalarials. These findings indicate that double mutations (1560 N plus 2874 T) are required for simultaneous resistance to high levels of MFQ, LUM, and PPQ. The NT mutations in the UBP1 had no significant effect on parasite responses to CQ, QN, and DHA.

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