reduces its overall activity. This idea is also
suggested by our finding that reduced activ-
ity of Kelch13 causes resistance (Fig. 4E). In
addition, previous work indicated that resist-
ant parasites harbor less Kelch13 protein ( 43 ).
In agreement with this observation, we found
~30 to 50% less Kelch13 in parasites with the
resistance-conferring form in Western blots
and by measuring Kelch13 levels directly in
living cells (Fig. 5, B to D). To test whetherincreased expression of Kelch13 can lead to
the loss of resistance, we episomally expressed
different versions of Kelch13 on the resistant
genomickelch13C580Ybackground. Adding
wtKelch13 (leading to expression of episomalBirnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 6of9
Fig. 4. A Kelch13-defined pathway in endocytosis and resistance.
(A) Fluorescence microscopy images of saponin-released young ring stages
(examples of spherical, partial, and full amoeboid parasite) grown for 3 to
6 hours in red blood cells preloaded with fluorescent dextran (Alexa647) show
internalized host cell cytosol (arrowheads). Scale bar, 5mm. (BtoD) Quantification
of dextran uptake into 3-to-6-hour rings after inactivation (rapa) of Kelch13
by KS in the 3xNLS line ( 17 ) (B) or KIC7 (C) compared with control (no rapa) or
in the K13mutparasites compared with wild type (3D7) (D). Each point shows the
amount of fluorescence internalized into one cell (arbitrary fluorescence units).
Data pooled from three independent experiments (individual experiments in
fig. S12) with a total ofn= 138, 140, 132, 130, 122, and 114 cells for Kelch13 KS
control, rapa, KIC7 control, KIC7 rapa, 3D7, and K13mut, respectively. Unpaired,
two-tailedttest.Pvalues and percent reduction of the mean are indicated.
(E) RSAs after inactivating Kelch13 by KS (rapa) compared with control with
Kelch13 3xNLS parasites ( 17 ). (F) RSAs with the TGD lines indicated. (G) Growth
rate of TGD lines. (H) RSAs after KS (rapa) to inactivate the proteins indicated
compared with control or in 3D7 after inhibiting growth using NaN 3 .(I) Heatmap
of transcription levels in 1-hour intervals across asexual blood stage development
[values from ( 42 )], clustered according to similarity for the proteins analyzed
in this study (see also fig. S13). Note that KIC2 was not part of the dataset.
Bottom shows data of colocalization, RSA, and endocytosis experiments (table S1;
Fig. 4, B, F, and H; and Fig. 3E; respectively) as heatmaps (color shading
indicated; gray, not applicable). Asterisk indicates ring-stage endocytosis assay.
(J) RSA with parasites harboring a mutatedubp1gene encoding a change
fromArgtoHisataminoacidposition3138( 19 ). Resistance in RSA defined
as mean survival above 1% [green line in (F), (H), and (J)], a previously set
standard ( 4 ). [(B) to (H), and (J)] Error bars show SD (not shown if close to
mean).Pvalues indicated [only selectedPvalues in (F) and (G)]; ns, not
significant. [(E) to (H), and (J)] Unpaired, two-tailedttest with Welch’s
correction. Each point indicates an independent experiment.RESEARCH | RESEARCH ARTICLE