Golgi trafficking and rab4 and 35 as representative

Golgi trafficking and Rab4 and 35 as representative GTPases
that regulate protein recycling from the plasma membrane.
Importantly, CT229:: bla L120D comp was unable to recruit
Rab GTPases to the vicinity of the inclusion ( Figure 2 F). Collec-
tively, our results highlight the importance of the CT229 SLD in
binding and recruiting Rab GTPases to the inclusion.
CT229 Targets Clathrin-Dependent Vesicle Transport
Pathways
The yeast suppressor screen is an innovative technique that we
( Weber et al., 2016a ) and others ( Guo et al., 2014; Tan and Luo,
2011; Tan et al., 2011 ) have used to identify host pathways
targeted by bacterial effector proteins. Many bacterial effector
Figure 2. Recruitment of Rab Effector Pro-
teins to the Chlamydial Inclusion Requires
CT229
HeLa cells were transfected with HA-OCRL or
GFP-RUFY1 and infected at an MOI of 1 for 18 h.
Cells were fixed with formaldehyde or methanol
and probed with anti-IncE (red) and anti-HA
(OCRL) antibodies. Scale bar represents 10 m m.
Data are representative of three independent
experiments.
Cell Reports 26 , 3380–3390, March 19, 2019 3383

proteins modulate essential cellular signaling pathways
(apoptosis, cytoskeletal function, vesicular traffic, etc.) and
when overexpressed in yeast, effector targeting of these path-
ways manifests as impaired growth. This phenotype can be ex-
ploited to identify the cognate host pathways that are targeted by
these effector proteins by conversely overexpressing a library of
host proteins to identify those that restore yeast growth. To iden-
tify the yeast pathway targeted, we co-expressed full-length
CT229 with a yeast genomic library. We identified 74 colonies
in which the growth defect was rescued; however, only 12 of
these consistently suppressed the growth defected associated
with expression of CT229. Sequencing of suppressor plasmids
Figure 3. Coiled-Coil SNARE-like Domain of CT229 Is Necessary for Chlamydia Replication and Rab Recruitment
(A) Bioinformatic analysis of CT229 identified a transmembrane domain (blue), a coiled-coil SLD (purple), and Y-based sorting signal (green).
(B) QuikChange mutagenesis was used to determine whether any of the predicted motifs are necessary for CT229 toxicity in yeast. CT229 variants were
transformed into S. cerevisiae W303 and spotted onto uracil dropout media to assess toxicity.
(C) To determine whether CT229 eukaryotic-like domains or the C terminus are required for CT229 binding to Rab GTPases, we introduced a single amino
acid substitution into the SLD (CT229
L120D
). CT229
FL
(full-length), CT229
D 197-215
, and CT229
L120D
were overexpressed as Flag-tagged fusions in C. trachomatis.
Flag-tagged CT229 was immunoprecipitated from HeLa cells transfected with Rab4 and samples were analyzed by western blotting. Data are representative of
three independent experiments.
(D) HeLa cells were infected at a MOI of 1; at 0, 24, and 48 h post-infection host cells were lysed; and infectious EBs were plated on new HeLa monolayers.