10 colocalizing proteins, eight also colocalized
in the resistance-relevant ring stage, whereas
the other two (KIC8 and KIC9) were not
detected in that stage (fig. S6 and table S1).
Thus DiQ-BioID identifies interactors and
compartment neighbors in living cells with
high specificity and in this study defined a
series of proteins proximal to the Kelch13-
Eps15 complex.Kelch13-Eps15 marks an AP-2 compartment
devoid of clathrin
Because Eps15 in other organisms is typically
involved in endocytosis ( 24 , 25 ), we hypothe-sized that the Kelch13 compartment might also
be involved in this process. In malaria blood
stages, endocytosis mediates the large-scale
uptake of host cell cytosol (consisting predom-
inantly of hemoglobin), but the molecular
details of this process are poorly understood ( 26 ).
In model organisms, the most common andBirnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 2of9
Fig. 1. Kelch13 location and interactome.(A) Fluorescence microscopy images
showing parasites with endogenously GFP-tagged Kelch13 (K13) ( 17 )with
coexpressed STVR-SDEL (ER), Sec13p (ER exit sites), P40 (phosphatidylinositol
3-phosphate sensor: food vacuole), ACP (apicoplast), or endogenously GFP-tagged
mutated Kelch13 (K13C580Y) with episomal mCherry-Kelch13wt. See fig. S1 for
all panels. (Bottom) Most common arrangement of markers in single-nucleus
trophozoites. (B) Scheme of DiQ-BioID. (C) Rapalog-dependent recruitment of the
biotinylizer (BirA*-FRB) to endogenously GFP- and FKBP-tagged Kelch13 (FKBP-
K13). (D) Top-right quadrant of scatter plot of Kelch13 DiQ-BioID, showing proteins
enriched (log 2 ratio) on rapalog (biotinylizer on target) compared with control
(biotinylizer cytoplasmic). Significantly enriched proteins are indicated (short, unique
names or PlasmoDB identifiers). False discovery rate (FDR) indicated for the least
significant experiment. Asterisks indicate proteins previously suspected in ART
resistance. Figure S2 shows full plot and replicas with a different biotinylizer.
(E) Fluorescence microscopy of parasites with endogenously 2xFKBP-GFP-2xFKBP–
tagged Eps15 episomally expressing mCherry-Kelch13. (F) Immunoprecipitation
(IP) of episomally expressed Kelch13 in parasites endogenously expressing
GFP-Eps15. I, input extract; P, post IP extract; W, last wash; E, eluate. Full blots
and replicas in fig. S4. (G) Top-right quadrant of scatter plot of Eps15 DiQ-BioID,
as in (D). Figure S5 shows full plot and independent replicas. (H)Fluorescence
microscopy in parasites with endogenously GFP-tagged DiQ-BioID hits (KIC1-10 or
short name) colocalized with episomal mCherry-Kelch13. Arrowheads indicate
Kelch13 foci overlapping with the KIC signal (all panels in fig. S6). Scale bars, 5mm.
Merge, merged green and red channel; DAPI, 4′,6-diamidino-2-phenylindole (nuclei).RESEARCH | RESEARCH ARTICLE