best studied form of endocytosis is mediated
by clathrin, which depends on the AP-2 adaptor
complex ( 27 , 28 ). AlthoughP. falciparum
Eps15 contains regions specific to malaria pa-
rasites, it shows typical hallmarks of Eps15 pro-
teins, including AP-2 binding sites (fig. S5B).
To test whether the Kelch13-Eps15 complex
marks an endocytic structure, we colocal-
ized Kelch13 with endogenously GFP-tagged
clathrin heavy chain (CHC) or AP-2m(fig. S3).
AP-2m–GFP foci colocalized with (Fig. 2A, ar-
rowhead) or were found in the same region
as Kelch13 foci (Fig. 2A, arrow), indicating
an endocytosis role for the Kelch13-Eps15 com-
partment. Unexpectedly, CHC was present in
foci that did not overlap with Kelch13 (Fig. 2A).
This showed that Kelch13 colocalizes with a
compartment that contains AP-2 but is devoid
of clathrin. Whereas clathrin typically has
additional functions besides endocytosis where
it associates with other adaptors, AP-2 is gen-
erally associated with clathrin-dependent en-
docytosis ( 27 , 28 ). AP-2 independent of clathrin
is a highly unusual configuration that, to
our knowledge, was so far only observed in
Aspergillus nidulans( 29 ). Of note, AP-2mhas
previously been suspected to be involved in
ART resistance ( 30 , 31 ).
To probe this distinction between clathrin
and the Kelch13-Eps15 complex and further
assess the specificity of the DiQ-BioID proce-
dure, we performed DiQ-BioID with the CHC
(fig. S7). This resulted in a list of high-confidence
hits differing from the Kelch13 and Eps15
DiQ-BioIDs and included known CHC inter-
actors such as the putative clathrin light chain
(even more enriched than the CHC itself) and
subunits of the AP-1 adaptor complex (Fig. 2B,
fig. S7, and data S1). Unexpectedly, subunits of
AP-4—in other organisms considered a clathrin-
independent adaptor of the trans-Golgi net-
work ( 32 , 33 )—were also enriched, whereasAP-3wasonlyverymildlyenriched,notreach-
ing statistical significance (data S1). AP-4 is
not well studied to date, but it is known to
interact with tepsin ( 34 , 35 ).P. falciparum
tepsin ( 34 ) was a prominent hit in the CHC
DiQ-BioID, validating the AP-4 hits. This sug-
gests that CHC associates with AP-4 in malaria
parasites, either indicating differences com-
pared to other organisms or that DiQ-BioID
uncovers interactions not retained using pre-
viously used approaches in other organisms.
Hence, DiQ-BioID reveals a credible CHC
interactome that is clearly distinct from the
Kelch13-Eps15 interactomes. The CHC DiQ-
BioID also detected a different Kelch protein
(PF3D7_1205400), potentially indicating that
different trafficking complexes harbor dis-
tinct Kelch proteins. Only PFK9, HSP90 (heat
shock protein 90), and FKBP35 were hits over-
lapping with the Kelch13 and Eps15 DiQ-BioID
experiments, suggesting that these hits mightBirnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 3of9
Fig. 2. Kelch13 complex locates with AP-2mbut is distinct to clathrin complex.
(A) Fluorescence microscopy images of parasites endogenously expressing 2xFKBP-GFP–tagged
clathrin heavy chain (CHC) or AP-2mwith episomal mCherry-Kelch13. Arrows mark overlap,
and arrowheads mark similar region of Kelch13 foci. Scale bar, 5mm. Merge, merged green
and red channel; DAPI (nuclei). (B) Top-right quadrant of scatter plot of CHC DiQ-BioID.
Figure S7 shows full plot and replicas with a different biotinylizer. (C) Heatmap of four Kelch13,
Eps15, and CHC DiQ-BioID experiments showing proteins enriched in at least three of four
experiments with FDR < 1%. K-means clustering into two clusters highlights overlap of
Kelch13 and Eps15 hits (dark green cluster) compared with CHC hits (light green clusters).
Gray blocks indicate not identified or no ratio due to missing label. Red font indicates DiQ-BioID
baits, and orange font indicates proteins further analyzed.
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