Fig. 1

AaCAT1 phylogeny and interaction screen. a AaCAT1 and AaCAT3 represent an expansion of the Drosophila Slif CAT family. Aedes aegypti CAT protein sequences were downloaded from the NCBI protein database, and Drosophila melanogaster (Dmel) CAT protein sequences were accessed from FlyBase version FB2021_04 [37]. Protein sequences were aligned using MEGA11 [38], and a neighbor-joining tree was generated in MEGA11 from the multiple sequence alignment. Numbers next to each node represent bootstrap values based on 500 replications. Both AaCAT1 and AaCAT3 have previously been annotated as Slif in Ae. aegypti. Our current assembly shows that both transporters share homology with D. melanogaster Slif and represent an expansion of the Slif transporter family during Ae. aegypti evolution. The red box around AaCAT1 denotes its use in our protein interaction assay. All gene names not preceded by Dmel are Ae. aegypti CAT annotations. Brackets around gene clusters represent our suggested re-annotations of Ae. aegypti CATs based on this tree. b Predicted transmembrane structure of AaCAT1. The Protter web-based application [40] was used to illustrate the 14 transmembrane helices. The substrate specificity site was marked [34], and the C-terminal 28-amino acid fragment used for bait in the Y2H assay is indicated by circles containing their single-letter amino acid code. c Table containing the top five interacting proteins with at least two distinct hits determined by Y2H assay. d AaCAT1 bait fragment for pull-downs and GCN1 interacting fragments. e Pull-down analysis of AaCAT1-GCN1 interaction. The AaCAT1 C-terminal domain was incubated with fat body proteins and the resultant complexes captured on cobalt resin. Coomassie blue staining of crosslinked (x-linked) and non-crosslinked (non) pull-down reactions using a 6-His-tagged AaCAT1 bait fragment (same as in d), and fat body protein reveals an eluted band at the expected size for GCN1 interaction (marked by arrow). f GCN1 binds to the C-terminus of AaCAT1. Western blot of pull-down assay fractions confirms presence of GCN1 in the pull-down elution fraction (marked by arrow). bait, Bait-only control; FT, flow-through fraction; elution, eluted fraction; PD, pull-down; resin, resin control; wash, resin wash fraction; Y2H, yeast two-hybrid; for other terms, see Abbreviations