Proteinase K protection assay shows that FP2a needs to be unfolded before crossing the PPM with its N-terminus first.

(A) Ring stage parasites were treated ± 10 nM WR99210 to inhibit the 120 aa FP2a reporter from unfolding. Trophozoite stage parasites were magnet purified and used for proteinase K (PK) protection assays. EQT was used to permeabilise the iRBC membrane, saponin (SAP) to lyse the PVM, and TX-100 to lyse all membranes. A 3.8 kDa shift was observed in the reporter size when the PV was lysed and treated with PK (lanes 4 and 9), which matches the predicted size of the 34 aa (4 kDa) N-terminus preceding TM domain (measured in Odyssey LI-COR). FP2a reporter appears to be more protected against PK degradation when treated with WR99210 vs non-treated, indicating trapping at the PPM not PVM. Chicken anti-FLAG was used to probe for the FP2a reporter, GBP130 is a soluble exported protein marker, SERA5 is a PV marker and GAPDH is a parasite cytoplasm marker. Blot is representative of 3 biological replicates, which showed a similar trend, SN = supernatant, P = pellet. Full-length blots are shown in S13 Fig. (B) The fold difference for protection of the FP2a reporter (FLAG) was measured by densitometry, where the FLAG signal was adjusted to GAPDH and the fold difference measured by adjusting the EQT + PK and SAP + PK signal to EQT + PBS. The fold difference (X) compared with untreated is indicated with a red dotted line on the graph. Error bars = SD from 3 biological replicates. (C) A suggested model derived from the PK assay results. When parasites were not treated with WR99210 more reporter cargo was degraded indicating that when inhibited from unfolding the reporter was trapped at the PPM, likely inhibiting extraction into the PV potentially facilitated by HSP101 as suggested for exported proteins with a transmembrane domain.