Science - USA (2020-01-03)

result from the method rather than being
biologically meaningful (Fig. 2, B and C). This
was supported by DiQ-BioID using the bio-
tinylizer alone (fig. S8 and data S2), and a
lack of colocalization with Kelch13 was experi-
mentally confirmed in the case of PFK9 (Fig.
1G and fig. S6). Therefore, we did not analyze
these proteins further.
Overall, these findings show that Kelch13
defines proteins at a compartment that colo-
calizes with the classical endocytosis adaptor
AP-2mbut that is entirely distinct from clath-
rin, which typically associates with the AP-2
complex in other organisms. This may indi-
cate the existence of an unusual clathrin-
independent endocytosis pathway in malaria
parasites.

Kelch13 compartment proteins are involved
in endocytosis

To investigate a possible role of the Kelch13-
Eps15 compartment in endocytosis, suggested
by the presence of Eps15 and colocation with
Ap-2m, we first carried out correlative light and
electron microscopy (CLEM) with the para-
sites expressing endogenously GFP-tagged
Eps15. This revealed that Eps15 resides in
proximity of host cell cytosol-filled membra-
nous structures in the parasite in three of three
analyzed cells and in five of five foci in these
cells (Fig. 3A and fig. S9, A to C). Hence, it can
be inferred that Kelch13, Eps15, AP-2, UBP1,
and the nine colocalizing KICs are present
proximal to host cell cytosol-filled structures
in the parasite, congruent with a role of the
Eps15-Kelch13 complex in endocytosis of host
cell cytosol. In support of this, we noted that in

90% of cells with vesicle-like structures visi-
ble in differential interference contrast (DIC),
Kelch13 foci were found at such structures (fig.
S9D), suggesting that these may represent the
host cell cytosol-filled structures observed by
CLEM and likely are cytostomes.
To more directly assess the function of KICs,
we first carried out a selection-linked integra-
tion gene disruption (SLI-TGD) screen ( 17 )to
establish which of the Kelch13 compartment
proteins are essential for parasite survival. Of
the 13 tested genes, only the ones encoding
Eps15, UBP1, and KIC7 were refractory to dis-
ruption and hence are likely essential for para-
site survival (Fig. 3B and fig. S3). Knock sideways
(KS) ( 17 ) to conditionally inactivate these pro-
teins, as well as AP-2m, confirmed their impor-
tance for parasite growth (Fig. 3, B and C,
and fig. S10). Note that the smaller effect on
growth after inactivating Eps15 likely is the re-
sult of inefficient KS (Fig. 3C and fig. S10). To
test whether Kelch13, UBP1, KIC7, Eps15, and
AP-2mare involved in endocytosis, we condi-
tionally inactivated them by KS (fig. S10) and
assessed hemoglobin uptake into the parasite
over 8 hours using a previously established
assay, which results in a bloated FV phenotype

ifendocytosisisoperational( 36 ). Inactivation

of UBP1, KIC7, Eps15, and AP-2msignificantly

reduced transport of hemoglobin to the FV,

whereas the controls or inactivation of Kelch13

or an unrelated essential protein [PF3D7_

0210200 ( 17 ), here called 2102] did not (Fig.

3D). Inactivation of KIC7, AP-2m,andtoalesser

extent Eps15, reduced growth over the 8-hour

assay time. However, the growth defect itself

was not the reason for the phenotype, as in-

ducing an unrelated growth defect using azide

did not impair endocytosis (Fig. 3E). Surpris-

ingly, inactivation of Kelch13 significantly

increased parasite size compared with the

control, and inactivation of UBP1 showed a

clear reduction of endocytosis but no signif-

icant reduction in cell size over the 8-hour

assay time (Fig. 3, D to F). We conclude that

proteins of the Kelch13 compartment play a

role in endocytic uptake of hemoglobin, but

Kelch13 itself does not appear to be necessary

for this process in trophozoites. The lack of

Birnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 4of9

Fig. 3. Kelch13 compartment proteins are involved in endocytosis of host cell cytosol.(A) CLEM

with a cell expressing endogenously tagged Eps15-GFP: confocal image (scale bar, 2mm), EM section and

magnification thereof (scale bars, 0.5mm). Arrowheads indicate vesicles containing material of host cell

density. N, nucleus; FV, food vacuole. Details and two more cells in fig. S9. (B) Summary of Kelch13

colocalization and SLI-TGD screen of DiQ-BioID hits. nd, not done. (C) Growth curves of parasites after

induction of KS. One of three independent experiments. Figure S10 shows replicas and details on KS.

(D) Example DIC images showing bloated (control) and nonbloated (inhibited endocytosis) FVs (partly

highlighted: dashed line and arrows) after KS (rapalog) and E64 treatment. (E) Quantification of number

of cells with bloated FVs after E64 treatment for control (no rapalog) and KS (rapa) for 8 hours (nfor

control/rapa: UPB1, 33/33; KIC7, 33/33; Kelch13, 32/33; AP-2m, 33/32; Eps15, 33/33; 2102, 33/33; 3D7

NaN 3 , 34/34). Fisher’s exact test. Replicas in fig. S10. (F) Growth (cell diameter) of cells scored for bloated

FVs after KS compared with control, data pooled from all three (five for NaN3) independent experiments,

with a total ofn= 99/97 cells for no rapalog/rapa with UBP1, 101/97 with KIC7, 134/134 with Eps15, 99/97 with

AP-2m, 100/100 with 2102, and 170/170 for 3D7 ± NaN 3. Unpaired, two-tailedttest. ns, not significant.

RESEARCH | RESEARCH ARTICLE