Fig. 1

Repeat organization, subcellular localization, and complex association of KPAF4. a Schematic repeat organization of kinetoplast polyadenylation factor 4 from Trypanosoma brucei (Tb) and Leishmania infantum (Li). Repeat boundaries were determined using the TPRpred online tool (https://toolkit.tuebingen.mpg.de/#/tools/tprpred) and adjusted according to Cheng et al. ¹⁷. Amino acids in positions 5 and 35/last potentially involved in adenosine recognition are indicated in separate columns. b Mitochondrial targeting of KPAF4-TAP fusion protein. Crude mitochondrial fraction was isolated by hypotonic lysis and differential centrifugation (crude mito), and further purified by renografin density gradient (pure mito). The latter preparation was extracted under conditions that separate matrix from membrane-bound proteins⁴⁵. Protein profiles were visualized by Sypro Ruby staining and KPAF4-TAP was detected with an antibody against the calmodulin-binding peptide. The mitochondrial enrichment was calculated by quantitative western blotting vs. total protein loading. Representative of two experiments is shown. c Tandem affinity purification of KPAF4. Final fraction was separated on 8–16% SDS gel and stained with Sypro Ruby. Representative of three experiments is shown. d KPAF4 co-purification with mRNA processing complexes. Fractions purified from parental cell line (beads, no tagged protein expressed), and mock and RNase-treated mitochondrial extracts were subjected to immunoblotting with antibodies against MERS1 NUDIX hydrolase (PPsome subunit), KPAP1 poly(A) polymerase, KPAF1 and KPAF3 polyadenylation factors, and GRBC1/2 (RNA editing substrate-binding complex, RESC) and RET1 TUTase (MPsome). Tagged KPAF4 was detected with antibody against calmodulin-binding peptide. RNA editing core complex (RECC) was detected by self-adenylation of REL1 and REL2 RNA ligases in the presence of [α-³²P]ATP. Representative of two experiments is shown. e Crude mitochondrial fraction was extracted with detergent and soluble contents were separated for 5 h at 178,000×g in a 10–30% glycerol gradient. Each fraction was resolved on 3–12% Bis–Tris native gel. Positions of native protein standards are denoted by arrows. KPAP1, KPAF4-TAP, MERS1, and GRBC1/2 were visualized by immunoblotting. REL1 and REL2 RNA ligases were detected by self-adenylation. Thyroglobulin (19S) and bacterial ribosomal subunits were used as apparent S-value standards. In each panel, representative of three experiments is shown