effect in trophozoites agrees with the previous
finding that inactivation of Kelch13 specifi-
cally affected the ring stage only ( 17 ).
A detailed comparison of the stage-specific
essentiality of Kelch13, UBP1, and KIC7 re-
vealed that all three proteins caused a sim-
ilar phenotype when inactivated in rings
(severe delay of ring stage with limited pro-
gression to the trophozoite stage). In contrast,
inactivation in early trophozoites prevented
development into schizonts in KIC7 and UBP1,
but not in Kelch13 (fig. S11). This suggests a
similar function of these proteins but that
Kelch13 is not essential for this function in
trophozoites. A previously established KS cell
line that only partially inactivates Kelch13
(Kelch13 3xNLS) and that does not noticeably
affect parasite growth in a 4-day growth assay
( 17 ) showed a mild delay of the ring stage (fig.
S11).Hence,whilenotlethal,partialinactiva-
tion of Kelch13 (3xNLS line) leads to a much
milder, but similar, effect compared with full
inactivation of Kelch13 (1xNLS line).
Kelch13 influences endocytosis and resistance
in ring stages
Hemoglobin by-products activate ART, and
decreased hemoglobin digestion reduces ART
susceptibility ( 37 – 39 ). Hence, our data might
indicate that reduced levels of endocytic up-
take of host cell cytosol could, by controlling
thesupplyofhemoglobinavailablefordiges-
tion and ART activation, be the mechanism of
ART resistance. However, this would need to
take place in the resistance-relevant ring stage
( 4 ).AsUBP1andKIC7affectedendocytosisin
trophozoites and showed a similar phenotype
in rings to Kelch13 (fig. S11), we reasoned that
Kelch13 might influence endocytosis specifi-
cally in ring stages. However, as it is not clear
whether young ring stages endocytose hemo-
globin at all ( 39 – 41 ), we decided to first clarify
this. Because of the overabundance of hemo-
globin in the parasite culture, we used host
cells loaded with fluorescent dextrans, a well-
established tool for studying endocytic uptake
in malaria parasites ( 36 , 40 ). These experi-
ments demonstrated uptake of host cell mate-
rial into young ring stages (Fig. 4A).
Next, we tested the role of Kelch13 in endo-
cytosis in 0-to-6-hour young rings [the time
the drug pulse is applied in ART susceptibility
assays ( 4 )] using the Kelch13 3xNLS parasites
(the cell line leading to only partial inactiva-
tion of Kelch13, to avoid detrimental effects on
parasite growth). Inactivation of Kelch13 sig-
nificantly reduced endocytic uptake in these
rings (Fig. 4B and fig. S12). Inactivation of
KIC7 similarly reduced endocytosis in rings
(Fig. 4C and fig. S12), congruent with its sim-
ilar KS phenotype to Kelch13 in rings and its
role in endocytosis in trophozoites (Fig. 3E).
Hence, KIC7 affects endocytosis in both rings
and trophozoites and, together with the sim-
ilar stage-specific phenotypes, suggests that
the Kelch13 compartment proteins are needed
for endocytosis in all asexual blood stages,
with the exception of Kelch13, which is only
needed for this process in rings.
To assess whether this could be the reason
for resistance, we next tested hemoglobin up-
take into rings of resistant parasites contain-
ing a mutated genomic Kelch13C580Y( 17 ). The
resistant parasites also displayed reduced endo-
cytic uptake (Fig. 4D and fig. S12), suggesting
this as a reason for reduced susceptibility to
ART. To further confirm this hypothesis, we
inactivated Kelch13 (again using the Kelch13
3xNLS line) and measured whether this can
render parasites ART-resistant using a standard
RSA ( 4 ). When Kelch13 was inactivated, para-
sites showed levels of resistance comparable
to the resistant Kelch13C580Yparasite line (Fig.
4E). Thus, Kelch13 inactivation reduces endo-
cytosis of host cell material and increases re-
sistance. We conclude that early ring stages
already endocytose hemoglobin, and that this
process is reduced in parasites with a resistance-
conferring mutation in Kelch13 or in which
Kelch13 is inactivated, and that this correlates
with ART resistance.Kelch13 compartment proteins are involved
in ART resistance
Having established that inactivation of Kelch13
induces ART resistance, we next assessed wheth-
er other Kelch13 compartment proteins also
play a role in ART resistance. First, we used
the cell lines with the disrupted, nonessential
KICs. Whereas parasites with disrupted KIC1,
2, 3, 6, 8, and 9, as well as KIC10 (which is not
Kelch13 compartment–associated), did not
show an altered response to ART, disruption of
KIC4, KIC5, and MCA-2 led to a reduced sus-
ceptibility in a standard RSA (Fig. 4F). While
the reduced susceptibility correlated with a low
growth rate of the KIC5 and MCA-2 disruption
lines (suggesting some importance of these
proteins for efficient parasite growth), there
was little correlation overall between growth
and ART resistance (Fig. 4G), excluding growth
levels as a factor in resistance. For instance,
the line with a disruption of KIC10, the only
non–Kelch13 compartment KIC (Fig. 1H), had
the third lowest growth rate but did not show
resistance (Fig. 4, F and G).
To assess the role of the essential proteins
AP-2m, Eps15, UBP1, and KIC7 in ART resist-
ance, we partially inactivated them (using KS)
before and during the ART drug pulse of the
RSA in a manner that did not kill the parasites.
Inactivation of KIC7, AP-2m, Eps15, and UBP1
induced ART resistance (Fig. 4H). In contrast,
reducing parasite viability with azide or inacti-
vation of an unrelated essential protein [2102
( 17 )] did not affect the outcome of the RSA,
indicating that the effect on ART susceptibility
is specific for the function of Kelch13 and itscompartment proteins (Fig. 4H). We conclude
that inactivation of more than half (7 of 13) of
the tested Kelch13 compartment proteins,
as well as Kelch13 itself, reduces the respon-
siveness to ART. Notably, all of the Kelch13
compartment proteins affecting endocytosis
(Fig. 3, D and E) also rendered parasites re-
sistant, establishing a strong link between he-
moglobin uptake and ART resistance (table S1).
We conclude that Kelch13 compartment pro-
teins are part of an ART resistance pathway,
and the correlation of reduced endocytosis and
resistance indicates that this is the mechanism
of this mode of resistance. To test whether the
corresponding genes belong to a coregulated
pathway, we assessed their stage-specific tran-
scription patterns across asexual blood stage
development ( 42 ). Except formca2, all genes
involved in resistance clustered into one group
when compared with (i) the other validated
Kelch13 compartment hits (Fig. 4I), (ii) all
significant Kelch13 interactome hits (fig. S13A),
and (iii) all hits significant in any of the BioIDs
(fig. S13B). In contrast, the other hits showed
widely differing transcription patterns, reflect-
ing the asynchronous nature of the parasites
used as input for the DiQ-BioID. Overall, the
coexpression lends independent support to
the idea that the genes experimentally iden-
tified here belong to a coregulated pathway
involved in hemoglobin endocytosis.
To show that the resistance pathway of the
Kelch13 interactors is clinically relevant, we
chose a mutation in UBP1 (R3138H) that was
identified as a possible contributor to ART re-
sistance by genomic surveillance of parasite
field samples ( 19 ). We changed this position in
UBP1 in 3D7 parasites and found that this ren-
dered these parasites resistant to ART (Fig. 4J)
at a level comparable to the resistance demon-
strated when UBP1 was inactivated by KS (Fig.
4H). These findings demonstrate that the path-
way identified here can be clinically relevant
and that mutations in the UBP1 can modulate
ART resistance.Reduced levels of Kelch13 explain
ART resistance
To determine how mutated Kelch13 influences
resistance, we first assessed whether the inter-
action profile of mutated Kelch13 was altered
compared with wtKelch13. However, DiQ-BioID
with the Kelch13C580Yparasite line revealed
no marked differences to the DiQ-BioID with
wtKelch13(Fig.5A,fig.S14,andtableS1).The
only exception was KIC10, which was less en-
riched in the Kelch13C580YDiQ-BioID, but this
is the only KIC not colocalizing with Kelch13
and has no role in resistance (Figs. 1H and 4F
and table S1). As these findings indicated that
there is no change in the interaction profile of
Kelch13C580Ycompared with wtKelch13, we
hypothesized that mutating Kelch13 does not
change a specific quality of its function butBirnbaumet al.,Science 367 ,51–59 (2020) 3 January 2020 5of9
RESEARCH | RESEARCH ARTICLE