Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum

Abstract

During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion) developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin) was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.

Figure 1. The retromer cargo-selective complex localizes to a novel, heritable subcellular compartment.

(A) Wide-field epifluorescence images of live parasites expressing PfVps35-YFP. Parasites are shown at ring (R), trophozoite (T), schizont (Sz), segmenter (Sg), and extracellular merozoite (M) stages. Hoechst 33342 fluorescence (DNA) is pseudocolored red. (B) Images of live parasites expressing PfVps29-YFP. (C) Images of live parasites co-expressing PfVps35-YFP and PfVps29-mCherry in trophozoite (T) and schizont (Sz) stages. mCherry (mC) fluorescence is pseudocolored red. (D) Co-localization of PfVps35-YFP or PfVps35-HA and organellar markers in fixed parasites (except for MitoTracker, which was imaged live). PMV, plasmepsin V; ACP, acyl carrier protein; MT, MitoTracker Red CM-H2Xros; RAP1, rhoptry associated protein 1; AMA1, apical membrane antigen 1. The AMA1 panel shows free merozoites; all others are intraerythrocytic. Organelles labeled by the markers are indicated in parenthesis. Marker-derived fluorescence is pseudocolored red. (E) PfVps35-YFP is adjacent to developing rhoptries in a 2N parasite. Hoechst 33342 fluorescence (DNA) is pseudocolored cyan. In all panels, YFP fluorescence is pseudocolored green. Scale bars, 2 µm.

Figure 2. PfRab7 localizes to the same compartment as the retromer cargo-selective complex.

(A) Effect of the DD-stabilizing agent trimethroprim (TMP) on the steady-state levels of DD-mCherry-PfRab7 in clonal parasite lines D9 (one expression cassette per genome) and G9 (two expression cassettes per genome). 3D7 is the untransfected parental parasite line. Upper: Anti-mCherry immunoblot. Lower: The membrane was reprobed with anti-glycerophosphoester phosphodiesterase (PfGDPD) antibodies for a loading control. The sizes of protein markers in kDa are indicated at left. (B) Images of DD-mCherry-PfRab7 in live clone G9 parasites. Stages shown are ring (R), trophozoite (T), schizont (Sz), and extracellular merozoite (M). (C) Live parasites expressing DD-mCherry-PfRab7ΔCC and DD-mCherry. (D) Live parasites co-expressing PfVps35-YFP and DD-mCherry-PfRab7. (E) Live parasites co-expressing PfVps35-YFP and DD-mCherry-PfRab6. (F) Live parasites expressing DD-mCherry-PfRab7 mutants. T22N and N125I are predicted to be “GDP-locked” and Q67L is predicted to be “GTP-locked”. 5′ UTR sequences driving expression are pfrab7 for N125I and Q67L and pfapp (aminopeptidase P) for T22N. In panels B, C and F, Hoechst 33342 fluorescence (DNA) is pseudocolored green and in panels D and E YFP fluorescence is pseudocolored green. In panels B–F, parasites were cultured in the presence of 10 µM trimethoprim. mCherry (mC) fluorescence is pseudocolored red. Scale bars, 2 µm.

Figure 3. PfRab6 but not PfRab7 or retromer rapidly redistributes to the ER upon brefeldin A treatment.

(A) Distributions of DD-mCherry-PfRab6, DD-mCherry-PfRab7 and PfVps35-YFP in live parasites after one hour in the presence of 5 µg/mL brefeldin A. Redistribution of DD-mCherry-PfRab6 to the perinuclear ER is indicated with an arrow (top panel). FP, fluorescent protein. mCherry fluorescence is pseudocolored red, YFP fluorescence is pseudocolored green and Hoechst 33342 fluorescence is pseudocolored cyan. Scale bar, 2 µm. (B) Effect of brefeldin A (5 µg/mL, 1 hour) on the percentage of parasites exhibiting ER-associated DD-mCherry-PfRab6 fluorescence. Results are an average of three experiments, n = 75 to 85 parasites per condition. The p-value was determined using a two-tailed Student's t-test. (C) Effect of Brefeldin A (5 µg/mL, 1 hour) on the number of puncta labeled with DD-mCherry-PfRab6, DD-mCherry-PfRab7 (clones D9 and G9) or PfVps35-YFP. Results are averages of three experiments, n = 50 to 70 parasites per condition.

Figure 4. The putative protein sorting receptor PfSortilin localizes to the P. falciparum Golgi apparatus.

(A) Schematic diagram of the domain organization of PfSortilin with the number of amino acids in each domain indicated below. At bottom is the C-terminal sequence of PfSortilin with the site of incorporation of the HA tag indicated. TM, transmembrane. (B) Anti-HA immunoblot of the membrane fraction of parasites expressing PfSortilin-HA (clone C9) and of the parental 3D7 parasite line. PfSortilin-HA is indicated with an arrow. Two cross-reacting species are present in both lanes (asterisks). The membrane was reprobed with anti-plasmepsin V (PMV) antibodies for a loading control. The sizes of protein markers in kDa are indicated at left. (C) Co-localization of PfSortilin-HA and ERD2 in aldehyde-fixed clone C9 parasites. T, trophozoite; Sz, schizont. Scale bar, 2 µm.