Identification of serine/arginine-rich splicing factor 1-interacting proteins in Plasmodium berghei

Abstract

Background

Serine/arginine-rich splicing factor 1 (SR1), an orthologue of hypothetical RNA-binding protein (HRB1) in yeast, is essential to the asexual development of Plasmodium falciparum and P. berghei. However, their interacting proteins in malaria parasites remain unclear.

Methods

To identify SR1-interacting proteins in malaria parasites, transgenic Pb ANKA expressing green fluorescent protein-fused PbSR1 (PBANKA_1232100) was generated and performed immunoprecipitation coupled to mass spectrometry (IP-MS) using the transgenic parasites. To investigate the developmental stage at which PbSR1 and RhopH2 are expressed, transgenic parasites co-expressing the fusion proteins PbSR1::GFP and RhopH2::mCherry were generated and western blot analysis and live-cell fluorescence imaging were performed.

Results

The fluorescence signal of PbSR1::GFP was stronger in nuclei than the cytoplasm of malaria parasites. IP-MS of the transgenic parasites indicated that PbSR1 interacts with nuclear proteins, including RNA-binding protein and small nuclear ribonucleoprotein, and cytoplasmic proteins, such as RhopH1 (or Clag3), RhopH2, and RhopH3. Live-cell fluorescence imaging showed that co-localization of PbSR1 and RhopH2 in the cytoplasm was observed in trophozoites and gametocytes but not mature schizonts and merozoites. From these results, trophozoites, immature schizonts and gametocytes are candidate stages at which cytoplasmic PbSR1 interacts with RhopH2.

Conclusions

PbSR1 interacts with nuclear proteins and rhoptry proteins.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12936-026-05786-3.

Introduction

Export of mRNA into the cytoplasm is essential to the survival of all eukaryotic cells. In opisthokonta, such as yeast and humans, the export receptor Mex67/Mtr2 plays a pivotal role in the terminal step of nuclear mRNA export [1, 2]. However, orthologues of Mex67/Mtr2 are absent from the genomes of Plasmodium, which are unicellular eukaryotes [3]. Thus, malaria parasites are considered to use an evolutionarily ancient mechanism that has been conserved throughout eukaryotic evolution [4].

Although orthologues of Mex67/Mtr2 are absent, malaria parasites possess all of the genes encoding adaptor proteins for Mex67/Mtr2, including nuclear poly(A) binding protein 2 (NAB2), yeast RNA annealing protein (YRA1), and three serine/arginine-rich (SR) proteins, including nucleolar protein 3 (NPL3), G-strand binding protein 2 (GBP2), and hypothetical RNA-binding protein (HRB1, SR1 in malaria parasites) [3, 4]. In yeast, deletion of NPL3, GBP2, and HRB1 does not affect cell growth [5]. However, SR1 has been shown to play essential roles in the asexual development of P. falciparum and P. berghei ANKA (Pb ANKA) [4, 6]. Moreover, the deletion of gbp2 affects sexual development [7].

SR1 derived from P. falciparum, designated PfSR1 (PF3D7_0517300), recognizes mRNAs containing specific sequence motifs [8]. However, PfSR1-interacting proteins in malaria parasites remain unclear. Elucidation of protein interactions provides important information about the role of RNA-binding proteins in malaria parasites [4, 9]. In the present study, screening of SR1-interacting proteins in malaria parasites using immunoprecipitation coupled to mass spectrometry (IP-MS) was performed. To date, proteins that interact with NAB2 and GBP2 in Pb ANKA [4] were identified by our previous studies. Moreover, RNA recognition motifs of SR1 in malaria parasites are highly conserved, showing 90% sequence identity between P. falciparum and Pb ANKA. Therefore, the SR1 derived from Pb ANKA, PbSR1 (PBANKA_1232100), was studied, and SR1-interacting proteins were investigated using transgenic Pb ANKA expressing green fluorescent protein (GFP)-fused PbSR1. IP-MS of PbSR1::GFP suggested that PbSR1 interacts with nuclear proteins and cytoplasmic proteins, especially RhopH2 (PBANKA_0830200). Live-cell fluorescence imaging showed that trophozoites, immature schizonts and gametocytes are candidate stages at which cytoplasmic PbSR1 interacts with RhopH2. From these results, a dual role for PbSR1 in the nucleus and cytoplasm in Pb ANKA was suggested.

Materials and methods

Mouse studies and ethics

Five- to six-week-old female C57BL/6J (B6) mice were purchased from CLEA Japan Inc. (Tokyo, Japan). The experiments were approved (#240823-7) by the Experimental Animal Ethics Committee of Azabu University (Kanagawa, Japan), and all experimental animals were kept at the animal facility in a specific-pathogen-free unit with sterile bedding, food, and water.

The infection studies included frequent observations to determine humane endpoints, at which mice were unable to ambulate sufficiently to obtain water or food. At the indicated time points, mice were euthanized by cervical dislocation under isoflurane. All experiments were designed to minimize suffering. No mice died before meeting the criteria for euthanasia. The investigators who conducted the experiments had completed the Experimental Animal Ethics Committee training course on animal care and handling.

Parasites and infection

Pb ANKA were stored as frozen stocks in liquid nitrogen. Erythrocytes parasitized with transfected parasites were generated in donor mice inoculated intraperitoneally with frozen stocks of parasites. The donor mice were monitored for parasitemia daily and bled for experimental infection during periods in which the level of parasitemia increased (1–2% parasitemia). Experimental mice were infected intravenously with 1 × 10⁴ parasitized erythrocytes or 5 × 10⁶ to 5 × 10⁷ purified mature schizonts harvested by Nycodenz density gradient centrifugation of infected blood from a given parasite strain.

Transfection

To generate transgenic parasites expressing GFP-fused PbSR1, the gene-targeting vectors for PbSR1 were designed and constructed as shown in Fig. S1. Specific primers annealing to either side of the green fluorescent protein gene (gfp)-mutated Plasmodium falciparum deoxyhypusine synthase (pfdhps)-expressing cassette [10] were used to amplified PCR fragments (Supplementary Table S1 and Fig. S1) as described previously [11, 12].

To generate transgenic parasites expressing mCherry-fused RhopH2, the gene-targeting vectors for RhopH2 were designed and constructed as shown in Fig. S1. Specific primers annealing to either side of the red fluorescent protein gene (mCherry)-hdhfr-expressing cassette were used to amplified PCR fragments (Supplementary Table S1 and Fig. S1) as described previously [11, 12].

The gene-targeting vectors were introduced into the 3' flanking regions of ORFs of target genes by double-crossover homologous recombination. Transfection was performed using an Amaxa Basic Parasite Nucleofector Kit (Amaxa GmbH, Cologne, Germany) according to the manufacturer’s protocol as described previously [13, 14].

PCR performed on genomic DNA

Genomic DNA of malaria parasites was extracted from peripheral blood of mice infected with malaria parasites using QIAamp DNA mini kit (Qiagen, Hilden, Germany). To generate gene-targeting vectors and confirm the introduction of gene-targeting vectors into target genes, PCR performed on genomic DNA was performed as described previously [13]. Thirty-five cycles of PCR were performed on a C1000 thermal cycler (Bio-Rad, Hercules, CA, USA). Each cycle consisted of denaturation at 98 °C for 15 s, annealing at 55 °C for 15 s, and extension at 68 °C for 1–6 min. The PCR products were then analyzed on a 1% (w/v) agarose gel and stained with ethidium bromide.

Parasitemia

Methanol-fixed tail-blood smears, stained with 3% Giemsa and diluted with phosphate buffer (pH 7.2) for 45 min, were subjected to microscopic examination. The number of parasitized erythrocytes (out of 250 erythrocytes) was enumerated when the level of parasitemia and gametocytemia exceeded 10%, while 1 × 10⁴ erythrocytes were examined in mice with lower levels of parasitemia and gametocytemia. The parasitemia and gametocytemia percentage values were calculated as follows: [(number of parasitized erythrocytes) ÷ (total number of erythrocytes)] × 100.

Fluorescence live-cell imaging

Parasitized erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N₂, 5% CO₂ and 5% O₂ at 37℃. Nuclear DNA was stained using Hoechst 33342 dye (Invitrogen, Waltham, MA, USA). To examine the localization of PbSR1::GFP and RhopH2::mCherry, Hoechst 33342 was added to the culture of parasitized erythrocytes at a concentration of 1 µg/mL. The staining medium was removed after incubation, and fresh RPMI1640 medium was added. Brightfield and fluorescence micrographs of blood smears were captured at 1000 × magnification using an All-in-One Fluorescence Microscope (BZ-X700; KEYENCE Japan, Osaka, Japan).

Protein immunoprecipitation (IP)

Parasitized erythrocytes were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N₂, 5% CO₂ and 5% O₂ at 37 ℃. Mature schizonts and gametocytes were harvested by Nycodenz density gradient centrifugation, as described previously [13]. Proteins were extracted using Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s protocol. Protein IP in transgenic parasites expressing PbSR1::GFP and transgenic parasites co-expressing PbSR1::GFP and RhopH2::mCherry was performed using GFP-Trap Agarose and a GFP-Trap-A kit, according to the manufacturer’s instructions (Chromotek, Planegg, Germany).

NanoLC-MS/MS analysis

All of the fractionated peptides obtained according to the manufacturer’s instructions of GFP-Trap-A kit were injected into a trap column (C18, 0.3 × 5 mm; L-column, Chemicals Evaluation and Research Institute, Tokyo, Japan) and an analytical column (C18, 0.075 × 120 mm; Nikkyo Technos, Tokyo, Japan), which was attached to a nano liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) system. The nanoLC-MS/MS analysis was conducted using an LTQ Orbitrap Velos mass spectrometer (Thermo Fisher Scientific) equipped with a nanoLC interface (KYA, Tokyo, Japan) and a nano high-performance liquid chromatography (nanoHPLC) system (DiNa; KYA). Purified peptides from the nanoLC were introduced into the LTQ Orbitrap Velos, a hybrid ion-trap Fourier transform mass spectrometer. Full MS and MS/MS scans were followed by higher energy collisionally activated dissociation (HCD). The database search engines Proteome Discoverer 1.4 (Thermo Scientific) and MASCOT 2.6 (Matrix Science) were used to identify and quantify proteins from the MS, MS/MS and reporter ion spectra of the peptides. Peptide mass data were matched by searching the protein database (PlasmoDB-59_PbergheiANKA.fasta), downloaded from PlasmoDB (updated August 22, 2022). The false discovery rate (FDR) [15] was calculated by peptide sequence analysis using Percolator software [16]. High-confidence peptide identifications were obtained by setting a target false discovery rate threshold of ≤ 1.0% at the peptide level. Proteins exhibiting at least three peptide spectral matches were included.

Western blot

Protein samples were electrophoretically separated on a Bis–Tris SDS-PAGE gel (4–12%, Cat. No. NP0323BOX, Invitrogen, Thermo Fisher Scientific Inc., Tokyo, Japan) using MES running buffer at 200 V for 22 min. After electrophoresis, the separated proteins were transferred onto a PVDF membrane with 0.2 µm pores (Cat. No. 1704156, Bio-Rad Laboratories, Inc., Tokyo, Japan) using the Trans-Blot Turbo Transfer System (Cat. No. 1704150J1, Bio-Rad Laboratories, Inc., Tokyo, Japan). The membrane was blocked with 5% dry skim milk (Cat. No. 4902720131292, Morinaga Milk Industry, Tokyo, Japan) in TBS-T (Tris-buffered saline containing 0.05% Tween-20, pH 7.4) for 1 h at room temperature with gentle shaking. The blocked membrane was then incubated overnight at 4 °C with rabbit anti-mCherry antibody (1:1000 dilution, Cat. No. 128508, GeneTex, Inc., CA, USA) diluted in TBS-T containing 1% dry skim milk. After washing with TBS-T, the membrane was incubated with goat anti-rabbit IgG-HRP (1:10,000 dilution, Cat. No. 5220-0458, SeraCare, MA, USA) in TBS-T containing 1% dry skim milk for 1 h at room temperature. Chemiluminescence was generated using Chemi-Lumi One Super (Cat. No. 02230-14, NACALAI TESQUE, Inc., Kyoto, Japan), and the signal was captured using the ChemiDoc Touch Imaging System (Cat. No. 1708370J1PC, Bio-Rad Laboratories, Inc., Tokyo, Japan). Following detection of the mCherry-fused Rhop2 protein, the membrane was incubated in antibody stripping buffer (Cat. No. T7135A, TaKaRa Bio Inc., Shiga, Japan) for 3 h at room temperature with gentle shaking. After several washes with TBS-T, the membrane was subjected to detection of GFP-fused SR1 using anti-GFP antibody (1:1000 dilution, Cat. No. 668205, BioLegend, CA, USA) as the primary antibody for 3 h at room temperature and rabbit anti-mouse IgG-HRP (1:5000 dilution, Cat. No. AP160P, Merck Millipore, MA, USA) as the secondary antibody for 1 h at room temperature.

Results

PbSR1 localizes to the nucleus and cytoplasm of malaria parasites

PbSR1 is essential to the asexual development of Pb ANKA [6]. In this study, to investigate PbSR1-interacting proteins by IP-MS, transgenic parasites expressing GFP-fused PbSR1 (PbSR1::GFP) were generated (Supplementary Table 1 and Fig. S1A). The GFP tag was introduced at the C terminus of endogenous PbSR1. PbSR1::GFP expression was controlled by the endogenous native promoters. Transgenic parasites were successfully generated (Supplementary Fig. S1A) that expressed the PbSR1::GFP fusion protein (Table 1 and Fig. 1). We first analyzed the subcellular localization of PbSR1::GFP. Live-cell fluorescence imaging revealed GFP signals in the erythrocytic stages (Fig. 1). In merozoites and mature schizonts, the fluorescence was localized in the nuclei of malaria parasites. Whereas, the fluorescence was localized to both the nuclei and cytoplasm of trophozoites, immature schizonts and gametocytes of malaria parasites (Fig. 1).

Table 1.

Results of immunoprecipitation coupled to mass spectrometry in PbSR1::GFP-expressing malaria parasites

Accession	Description	# AAs	MW [kDa]	calc. pI	Wild type				SR1::GFP-1
					Score	Coverage	# Peptides	# PSM	Score	Coverage	# Peptides	# PSM
PBANKA_0830200	High molecular weight rhoptry protein 2	1354	159.9	8.00	1755.25	28.66	27	76	23,357.07	43.21	50	718
PBANKA_1232100	Serine/arginine-rich splicing factor 1, putative	315	36.3	11.00	1351.89	27.94	8	41	26,459.41	35.87	14	688
PBANKA_1400600	Cytoadherence linked asexual protein, putative	1296	152.1	8.53	594.03	18.13	20	31	15,218.38	48.23	54	549
PBANKA_0416000	High molecular weight rhoptry protein 3, putative	908	105.0	6.92	680.32	16.19	15	31	7690.82	40.75	35	295
PBANKA_0931200	Heat shock protein 101	907	103.1	8.97	359.99	10.25	8	15	6267.26	49.83	39	197
PBANKA_1032100	Rhoptry-associated protein 1	604	70.6	8.06	595.50	22.52	10	23	5294.85	46.03	24	155
PBANKA_1464900	Rhoptry neck protein 3, putative	2248	264.7	8.92					3975.37	28.96	49	135
PBANKA_0912100	Casein kinase 1, putative	323	37.6	9.29	143.64	15.17	4	7	5933.47	44.27	15	169
PBANKA_1405400	U1 small nuclear ribonucleoprotein A, putative	468	53.9	8.53					5255.26	49.15	16	159
PBANKA_1101400	Rhoptry-associated protein 2/3	388	45.7	8.56	310.45	28.35	10	18	3636.51	48.20	17	121
PBANKA_0108700	RNA-binding protein 25, putative	679	80.5	5.58	25.60	2.36	1	1	4462.20	46.54	27	148
PBANKA_1415000	Pre-mRNA-processing factor 40, putative	789	93.4	8.54	129.57	6.21	4	5	4148.55	28.52	20	125

Proteins were extracted from PbSR1::GFP schizontand gametocyte-enriched cultures after culturing for 22 h. The top 12 proteins with fold change ≥ 5 relative to the controls are listed. Controls experiments corresponding to immunoprecipitations performed with wild-type Pb ANKA using anti-GFP beads coupled to mass spectrometry. Experiment was performed using one clone of wild-type Pb ANKA and three independent clones (SR1::GFP-1, SR1::GFP-2, and SR1::GFP-3) of SR1::GFP-expressing parasites (see Table S2). Representative data are shown. Abbreviations: PSMs, peptide spectrum matches; AAs, amino acids; MW, molecular weight; calc. pI, calculated isoelectric point.

Fig. 1.

Cellular localization of PbSR1::GFP-expressing malaria parasites. Erythrocytes parasitized by transgenic malaria parasites expressing PbSR1::GFP (green) were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N₂, 5% CO₂ and 5% O₂ at 37℃. The nuclei were stained with Hoechst 33342 (Hoechst, blue). Gametocytes were characterized by the size of the parasite, the presence of a single nucleus and nuclear enlargement. At least 50 parasitized erythrocytes were analyzed, and the same fluorescence pattern was observed in all parasitized erythrocytes. Representative data are shown. Scale bar = 5 µm

Identification of PbSR1-interacting proteins in malaria parasites

Next, protein IP using anti-GFP beads was performed, and the proteins bound to PbSR1 were identified using MS. IP-MS was performed using anti-GFP beads in wild-type Pb ANKA as a control. In three independent comparative proteomics analyses, 805, 862, and 914 proteins were detected. Among them, 187 proteins with at least 20 peptide spectral matches (the sum obtained from three independent experiments) and fold change ≥ 5 relative to the control in three independent experiments were analyzed further (Table 1 and Supplementary Table S2). Among the top-ranking proteins that bound to PbSR1 were rhoptry proteins, such as high molecular weight rhoptry protein 2 (RhopH2, PBANKA_0830200); cytoadherence linked asexual protein (RhopH1A or Clag3, PBANKA_1400600), RhopH3 (PBANKA_0416000); and rhoptry-associated protein 1 (RAP1, PBANKA_1032100) and RAP2/3 (PBANKA_1101400; Table 1).

Through IP-MS of PbSR1::GFP, nuclear proteins including RNA-binding protein (PBANKA_0108700, PBANKA_1202700), small nuclear ribonucleoprotein (PBANKA_1405400, PBANKA_1143000, PBANKA_1363000, PBANKA_0708400), and ATP-dependent RNA helicase UAP56 (PBANKA_0306800) were also detected (Table 1 and Supplementary Table S2). These results suggest that PbSR1 interacts with nuclear and cytoplasmic proteins.

Western blot analysis of IP lysate from parasites co-expressing PbSR1::GFP and RhopH2::mCherry using anti-GFP beads

IP-MS of PbSR1::GFP identified RhopH2 as a candidate protein for PbSR1 binding (Table 1). To confirm whether PbSR1 binds full length of RhopH2, transgenic parasites co-expressing the fusion proteins PbSR1::GFP and RhopH2::mCherry were generated (Supplementary Fig. S1B). The mCherry tags were introduced at the C terminus of endogenous rhoph2. rhoph2::mCherry expression was controlled by the endogenous native promoters. Transgenic parasites were successfully generated (Supplementary Fig. S1B). Western blot analysis showed that full length of PbSR1 were obtained using anti-GFP beads (Fig. 2A). As shown in Fig. 2B, full length of RhopH2 was detected in the IP lysate anti-GFP beads. These results suggest that PbSR1 binds full length of RhopH2.

Fig. 2.

Western blot analysis after IP with anti-GFP beads. A Western blot analysis with anti-GFP antibody. The molecular weight of the PbSR1::GFP fusion protein is approximately 63.2 kDa, consisting of 36.3 kDa (PbSR1) and 26.9 kDa (GFP) components. B Western blot analysis with anti-mCherry antibody. The molecular weight of the RhopH2::mCherry fusion protein is approximately 186.6 kDa, consisting of 159.9 kDa (RhopH2) and 26.7 kDa (mCherry) components. Note; The signal at approximately 25 kDa is likely due to a digested mCherry tag produced during sample preparation. The experiment was performed using one clone of parasites expressing PbSR1::GFP (SR1) and three independent clones (IP1, IP2 and IP3) of parasites co-expressing PbSR1::GFP and RhopH2::mCherry (RhopH2). Total; 35 µL of lysate (total protein) before immunoprecipitation using anti-GFP beads. The lysate was obtained from parasites co-expressing PbSR1::GFP and RhopH2::mCherry incubated for 22 h. IP; 5 µL of lysate after immunoprecipitation using anti-GFP beads

Live-cell fluorescence imaging of transgenic parasites co-expressing the fusion proteins PbSR1::GFP and RhopH2::mCherry

To examine the developmental stage at which PbSR1::GFP and RhopH2::mCherry express, live-cell fluorescence imaging was performed. As shown in Fig. 3, the dotted mCherry signal was present in the cytoplasm and was not co-localized with GFP signals in merozoites (Fig. 3). In trophozoites and gametocytes, the diffused mCherry signals were presented with GFP signals in the cytoplasm (Fig. 3). In immature schizonts, dotted and diffused signals were observed and diffused signals were presented with GFP signals in the cytoplasm (Fig. 3). In mature schizonts, dots of mCherry signals were detected in the cytoplasm but their signals were not co-localized with GFP signals (Fig. 3). In wild-type Pb ANKA, no GFP or mCherry signals were observed in all parasitized erythrocytes (Supplementary Fig. S2). These findings suggest that RhopH2 were presented with PbSR1 in the cytoplasm of trophozoites, immature schizonts and gametocytes.

Fig. 3.

Live-cell fluorescence imaging of PbSR1 and RhopH2 in the malaria parasites. Erythrocytes parasitized by malaria parasites co-expressing PbSR1::GFP (green) and RhopH2::mCherry (red) were transferred to RPMI1640 medium supplemented with 25% fetal bovine serum, 0.05 mg/mL penicillin and 0.05 mg/mL streptomycin. The parasitized erythrocytes were incubated for 22 h in 90% N₂, 5% CO₂ and 5% O₂ at 37℃. The nuclei were stained with Hoechst 33342 (Hoechst, blue). Gametocytes were characterized by the size of the parasite, the presence of a single nucleus and nuclear enlargement. At least 50 parasitized erythrocytes were analyzed, and the same fluorescence pattern was observed in all parasitized erythrocytes. Representative data are shown. Scale bar = 5 µm

Discussion

In this study, PbSR1-interacting proteins were identified using transgenic parasites expressing PbSR1::GFP fusion proteins. Results of IP-MS suggested that PbSR1 interacts with nuclear proteins and cytoplasmic proteins. These results suggest a possibility that PbSR1 plays differing roles in the nucleus and cytoplasm.

IP-MS of PbSR1::GFP showed that PbSR1 interacts with nuclear proteins. Some of these nuclear proteins have been found to interact with PbGBP2 and PbNAB2, which are involved in mRNA export [4]. These findings suggest that PbSR1 is involved in mRNA export in malaria parasites. PfSR1, orthologues of PbSR1, has recognized mRNAs containing specific sequence motifs [8] and exported to the cytoplasm from nucleus [3]. However, the detailed mechanism through which SR1 exports mRNA to the cytoplasm from the nucleus remains unclear, as no nuclear pore complex proteins or export receptor-like proteins were detected in IP-MS of PbSR1::GFP.

In a previous study, it has been shown that PbNAB2 interacts with nuclear pore complex proteins and is exported to the cytoplasm [4]. Moreover, NAB2 from the cytoplasm was imported into the nucleus by transportin [4]. On the other hand, the results of IP-MS showed that PbSR1 and PbGBP2 do not interact with transportin. PbGBP2 localizes nucleus and cytoplasm [4]. The cytoplasmic PbGBP2 causes translational repression during gametocyte development through interactions with ALBA4, DOZI, and CITH [4], and deletion of gbp2 affects the development of gametocytes [7]. Meanwhile, the results of IP-MS indicated interaction of PbSR1 with cytoplasmic proteins, such as RhopH1 (or Clag3), RhopH2, and RhopH3, in the cytoplasm. These results suggest that the role of cytoplasmic PbSR1 differs from that in nuclei.

The RhopH complex consists of three proteins, namely Clag3, RhopH2, and RhopH3, and is transported into the rhoptries of merozoites [17–21] and contributes to the development of the parasitophorous vacuole [22, 23]. The RhopH complex is involved in channel-mediated nutrient uptake. Specifically, RhopH3, but not Clag3 or RhopH2, has been shown to play a role in merozoite invasion [24–26]. Live-cell fluorescence imaging showed that the dots of signals of RhopH2::mCherry were not co-localized with GFP signals in merozoites and mature schizonts. These results suggest that PbSR1 does not interact with RhopH2 which is trafficked in the rhoptries.

RhopH2 and RhopH3 are synthesized in the cytoplasm of late trophozoite stage of P. falciparum [17, 26]. Live-cell fluorescence imaging showed that diffused signals of RhopH2::mCherry and PbSR1::GFP were observed in the cytoplasm of trophozoites, inmature schizonts and gametocytes, suggesting that trophozoites, immature schizonts and gametocytes are candidate stages at which cytoplasmic PbSR1 interacts with untransported RhopH2. As the period of differentiation into the schizont from the late trophozoite is a few hours, it is considered that regulation of synthesized rhoptry proteins is necessary for transport soon after differentiation into the schizont. Based on our findings, SR1 might be associated with stage-specific post-translational modifications or quality control for untransported rhoptry proteins, in the late trophozoite and immature schizont. In addition, transcription of rhopH and clag genes has been observed in gametocyte of P. falciparum [27]. Cytoplasmic SR1 might be also involved in differentiation into gametocyte.

Conclusion

In this study, a dual role for PbSR1 in the nucleus and cytoplasm in Pb ANKA was suggested. However, this possibility relies solely on IP-MS, western blot and co-localization data, which is inadequate to demonstrate direct interaction or functional causality. In future study, in vitro binding assay with recombinant proteins should be performed to validate the interaction between PbSR1 and PbRhopH2 proteins observed after immnoprecipitation. On the other hand, deletion of HRB1 does not affect growth in yeast [5]. The dual roles of PbSR1 in the nucleus and cytoplasm in asexual blood-stage parasites suggest it as a potential target for developing new drug.

Author contributions

M.N. designed research; M.N., Y.K., Y.F. and T.F. performed research; M.N., Y.K., Y.F., K.K. and T.F. analyzed data; and M.N., Y.K., Y.F., K.K. and T.F. wrote the paper.

Funding

This work was supported by a Grant-in-Aid for Scientific Research (C) from JSPS (No. 21K06997 and 24K10194) to M.N. This work was also supported in part by a Grant-in-Aid from the Institute for Fermentation (G-2025-2-027) to Y.F. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability

All data generated or analyzed during this study are included in this published article and its supplementary information files.

Declarations

Ethics approval and consent to participate

The experiments were approved by the Experimental Animal Ethics Committee of Azabu University, Kanagawa.

Consent for publication

All contributing authors agreed to consent for publication of the manuscript by Malaria Journal.

Competing interests

The authors declare no competing interests.